Effect of Altitude on Polyphenol Content, Antioxidant Activity and Multi-Element Composition of Wildflower Honey

Abstract

The aim of this study was to evaluate the influence of altitude on the phenolic content, antioxidant activity and mineral content of multifloral honeys collected in three different areas (plain, hill and mountain areas) of the Basilicata region. Our results show that the total phenolic and flavonoid contents and the multimineral profile were influenced by the altitudes of the different agro-climatic areas which are characterized by different soil characteristics and floral biodiversity. There was a negative correlation between altitude and total phenols, but there was a positive correlation between altitude and total flavonoids. Furthermore, altitude is closely related to antioxidant activities. Furthermore, the low correlations between antioxidant activities and polyphenols indicate that antioxidant activity is not only promoted by polyphenols but also by other biologically active substances (catalase, ascorbic acid and proteins) which contribute to the antioxidant activity of honey. This research demonstrates how different altitudes influence the analyzed parameters, confirming the uniqueness of honey with respect to the area of origin.

1. Introduction

Honey, a sugary substance produced by Apis millifera, has always attracted great interest for its exceptional nutritional value and its countless therapeutic and cosmetic uses [1]. Its chemical composition is strongly influenced by the geographical area, the climatic conditions and the biodiversity of the flora present in the bees’ grazing area [2]. A very important characteristic is that if the same floral and vegetal species are present in different geographical positions, the composition of honey varies considerably [3]. This happens because the abundance of phytochemicals depends on environmental conditions such as light, soil, humidity and temperature [3]. The Basilicata region, located in the south of the Italian peninsula, is characterized by pedoclimatic heterogeneity and different weather conditions; therefore, it has the right requirements for the development of beekeeping, and in fact, it hosts various pastures of honey bees. Honey is mainly composed of sugars (25–45% of fructose and 20–40% of glucose), but it is characterized by an abundance of components including amino acids, enzymes, proteins, vitamins, minerals, ash, organic acids and phenolic and flavonoid compounds that contribute significantly to its biological activity [4]. It is known that the botanical origin both influences the characteristics of honey [5] and determines its category for marketing [6]. Multifloral honey is characterized by its richness in floral combinations; in fact, it does not come from a single flower but from a group of flowers present within a 5 km radius of the hives [7]. Many scientific reports have demonstrated a strong antioxidant power of honey thanks to its bioactive components [8]. Furthermore, phenol and flavonoid compounds have anti-inflammatory [9], antibacterial [10] and antidiabetic effects [11]. However, the concentrations of phenolic substances vary from honey to honey since the botanical origin, the vegetal source and the geographical position considerably influence their contents [12]. However, the composition and nutraceutical characteristics are strongly influenced by altitude, which determines ecological and geographical changes. In the literature, there are some studies that have investigated the effect of altitude on the biological compositions of plants, but there is very little research on the effect of altitude on the mineral content and biological composition of Italian honey. Some African [13] and Indian [14] researchers have investigated the impact of the different altitudes of their country on the composition of honey, highlighting how altitude positively influences the content of polyphenols. Furthermore, it has been clarified that the content of polyphenols could vary both from plant sources and from the geographical origin of honey; consequently, their concentrations vary significantly between types of honey and locations [15,16]. The main objectives of this research were to evaluate the influence of altitude on the phenolic content, antioxidant activity and mineral content of multifloral honeys collected in three different areas (plain, hill and mountain areas) of the Basilicata region.

2. Materials and Methods

2.1. Sample Collection

The multifloral honey samples were collected from three different agro-climatic zones of the Basilicata region in 2023. The identified zones were plain (from 0 to 400 m a.s.l.), hill (from 600 m to 800 m a.s.l.) and mountain (from 900 to 1080 m a.s.l.) zones. Within each area, multifloral honeys were collected (n = 14) from apiaries inhabited by bees that grazed in the reference area. The honeys were filtered to remove impurities and subsequently packaged in sterilized glass jars and stored at room temperature until analysis.

2.2. Preparation of Honey Samples

All honey samples were prepared to extract the total phenols and flavonoids and antioxidant activities. The method described by Özkök et al. [17] was followed with minor modifications. An aliquot of honey was mixed with 75% (v/v) ethanol/water, homogenized and sonicated in an ultrasonic bath (US) (Elma Transsonic 460/H, Singen, Germany) for 30 min at room temperature and finally centrifuged.

2.3. Total Phenolic Content (TPC)

Total phenolic content (TPC) of honey trucks was determined using the Folin–Ciocalteu method, as suggested by Singleton et al. [18]. An aliquot of the ethanolic supernatant was added to 150 µL of Folin–Ciocalteu reagent (Sigma-Aldrich, Milan, Italy) and 3000 µL of 7.5% (w/v) sodium carbonate. The samples were placed at room temperature in the dark for 1 h. The absorbances of the samples were read at 760 nm against the blank (distilled water instead of the sample). Total phenol content was estimated by constructing a calibration curve with gallic acid (Sigma-Aldrich, Milan, Italy) at known concentrations (from 5 to 700 µg/mL). The results were expressed as mg GAE/100 g of honey.

2.4. Total Flavonoid Content (TFC)

The total flavonoid content (TFC) was estimated according to the method suggested by Meda et al. [19] with minor modifications. Briefly, 150 µL of ethanolic extract was mixed with 1500 µL of 2% AlCl₃ methanol and 1350 µL of methanol. The samples were stored at room temperature for 30 min and read at 415 nm. Each sample had its own blank consisting of the sample and methanol only (without 2% AlCl₃; w/v). Known concentrations of quercetin (5 to 600 µg/mL; Sigma-Aldrich, Milan, Italy) were used to calculate the total flavonoid content, expressed as mg QUE/100 g honey.

2.5. ABTS

The antioxidant activity of honey samples was estimated using the methods of Re et al. [20]. Briefly, an aliquot (100 μL) was added to 3900 μL of ABTS solution (0.700 ± 0.05 nm) and stored in the dark for 15 min. The absorbance of the samples and the Trolox standard (50–750 μg/mL) was read at 734 nm. The results were expressed in mg Trolox equivalents/g honey.

2.6. FRAP

The antioxidant activity of FRAP was performed using the method described by Ahmed et al. [21]. The FRAP assay estimates the reduction of ferric triridyltriazine [FeIII(TPTZ)] (colorless) to ferrous triridyltriazine [FeII(TPTZ)] (blue color). The FRAP reagent consisted of 10 mL of 0.3 M acetate buffer, 1 mL of 0.01 M TPTZ in 0.04 M HCl and 1 mL 0.02 M FeCl₆H₂O. The mixture was kept at 37 °C until analysis. An aliquot of supernatant (200 µL) was added to 2800 µL of FRAP. Samples were read at 593 nm against a blank (distilled water in place of sample). A calibration curve was made at known concentrations of Trolox (25–600 µg/mL). The results were expressed as µmol Trolox/g honey.

2.7. Determination of Element Content

In this research study, a validated approach for the simultaneous determination of 26 elements was applied, analyzing 7 oligoelements (Ca, Mg, Cu, Fe, Mn, Na and Zn) and 19 trace elements (Ag, Al, As, Ba, Be, Cd, Co, Cr, Hg, Li, Mo, Ni, Pb, Sb, Se, Sn, Sr, Ti and V) by using inductively coupled plasma mass spectrometry (ICP-MS) after microwave digestion. An amount of each honey sample (ca 50 g) was heated to 40 °C for 30 min in a water bath and stirred using a glass stick. The digestion procedure based on the UNI EN 13805:2014 reference method (European Committee for Standardization, 2014 [6]) was applied as follows: about 0.5 g (with a precision of 0.1 mg) of homogenized sample was weighed using an analytical balance (Mettler Toledo, Novate Milanese, Milan, Italy) and placed into a Teflon vessel, and then 8.5 mL of 68% (v/v) HNO₃ and 1.5 mL of 30% (v/v) H₂O₂ were added. The digestion procedure was performed by using a microwave system (Ethos-Easy Microwave Reaction System, Milestone, Sorisole, Bergamo, Italy) with the following time–temperature program: heating to 120 °C in 20 min and remaining at a constant temperature for 15 min; heating to 200 °C in 15 min and remaining at a constant temperature for 20 min; and a final cooling stage (30 min) to reach room temperature. The solution was transferred into 50 mL polypropylene disposable tubes and diluted with ultrapure water to a volume of 50 mL. This final solution was used for elemental analysis by ICP-MS.

2.8. Analysis by ICP-MS

An inductively coupled plasma mass spectrometer (ICP-MS, NexIon 2000 PerkinElmer, Waltham, MA, USA) equipped with a concentric nebulizer, a cyclonic spray chamber and a quartz torch was used. The operational parameters were as follows: 1600 Watt for radio frequency, 15 L/min for plasma gas (Ar), 1.0 L/min for nebulizer gas (Ar), a 50 ms dwell time and 20 sweeps/reading. Rhodium and bismuth added to standard and sample solution by on-line mixing were used as internal standards. The following elements/isotopes were detected: ¹⁰⁷Ag, ²⁷Al, ⁷⁵As, ¹³⁸Ba, ⁸Be, ¹¹¹Cd, ⁵⁹Co, ⁵²Cr, ⁶³Cu, ⁵⁶Fe, ²⁰²Hg, ⁷Li, ⁵⁵Mn, ⁹⁸Mo, ⁶⁰Ni, ¹²⁰Sb, ⁷⁸Se, ¹¹⁸Sn, ⁸⁸Sr, ⁴⁸Ti, ⁵¹V and ⁶⁶Zn. The sum of ²⁰⁶Pb, ²⁰⁷Pb and ²⁰⁸Pb were counted together to improve signal sensitivity. To minimize isobaric interference, the kinetic energy discrimination (KED) system was used, employing helium gas (99.9999%) at 3.8 mL/min (Low KED flow) for the determination of Co, Cu, Mn, Se and V, while the rate of 4.8 mL/min (High KED flow) was used for the determination of Al, Cr, Fe and Ti. Matrix effects were cut down by standard additions into the mineralized solution: for each element, 5 different addition levels were used, including the zero level. The addition levels, stated on the basis of a previous semi-quantitative analytical method by ICP-MS, were as follows: Ag, As, Be, Cd, Co, Hg, Li, Mo, Sb, Sn, Ti and V (0.40–2.0–10–40 µg/kg); Ba, Cr, Li, Ni, Pb, Se and Sr (4.0–20–100–400 µg/kg); Cu (20–100–500–2000 µg/kg); and Al, Fe and Mn (40–200–1000–4000 µg/kg). The major trace elements Ca, Mg and Na were determined using a semi-quantitative analytical tool using two calibration levels (5.0 and 50.0 mg/kg).

For all analytes, the calibration curves showed determination coefficients (R²) higher than 0.999. The limits of quantification (LOQs) were calculated as 10 times the standard deviation of 20 blank replicates for each element. All samples were analyzed in duplicate. Repeatability lower than 15% was observed for the results. The accuracy of the methods was checked by spiking experiments into a representative honey sample. Recovery factors ranging from 0.91 to 1.05 were obtained, and no correction factor was applied to the results.

2.9. Statistical Analysis

Statistical analysis was performed using the general linear model (GLM) procedure of a statistical analysis system (SAS Institute, Cary, NC, USA, 1996) using a monofactorial model. The Tukey post hoc test was used for a comparison of means, and differences were considered significant (p < 0.05). Results are presented as mean ± standard deviation (SD). Pearson’s correlation test was conducted to determine the linear correlation between the variables. The correlation coefficient (r) was also calculated.

3. Results and Discussions

3.1. Total Phenols, Flavonoids and Antioxidant Activity in Multiflora Honeys

The total phenolic (TPC) and flavonoid components (TFC) of honey are present in small concentrations and derive from the pollen of the pinatas that bees feed on. In Figure 1, the average content of polyphenols and flavonoids of the multifloral honeys studied is shown. The average content of phenols was 45.85 ± 6.61 mg GAE/100 g of honey, while the average value of total flavonoids was 4.35 ± 0.78 mg QUE/100 g of honey. The average content of polyphenols was similar to that found in Italian honeys by Pichichero et al. [22], but it was higher than that found by Habryka et al. [23] in Polish multifloral honeys (30.8 mg GAE/100 g). Considering the total phenolic content, our samples showed a higher content than multifloral honeys from northern Belgium (36.6 ± 1.78 mg GAE/100) [24]. Higher values were found by Nascimento et al. [25] in Brazilian honeys (100 mg GAE/100 g), and similar values to ours were reported by Bertoncelj et al. [26] in Slovenian honeys. The total flavonoid content of our samples was in line with those of Algerian [27] and Estonian [28] honeys, while it was lower than those of Polish [29] and Argentinian [30] honeys and higher than that of Indian honeys [31]. The variability found among honey samples could be related to the different environmental conditions, specific for each country [14]. Temperature, humidity, soil type, irradiation, climate trend and biodiversity of pastures certainly influenced the polyphenol content [2]. A statistical analysis showed that the altimetric zone significantly influenced the contents of total phenolics and total flavonoids (p < 0.001). High variability was observed among honeys from different areas studied, and significant differences (p < 0.001) were found in the mean TPC and TFC values among multifloral honeys collected from plain, hill and mountain areas. These results are consistent with another scientific report that observed a TPC ranging from 345.1 to 502.1 mg GAE/kg in multifloral honey from cerana bee (Apis cerana) collected from some regions of China [32]. Meanwhile, honey from different locations in Burkina Faso contained different TPC (32.59–100.39 mg GAE/100 g) and TFC values (0.41–8.35 mg QUE/100 mg) [19], related to the different floral types, the geographical origin of the honey, the climatic conditions and the conditions of production and conservation of the honey [33]. In the comparison between honeys collected at different altitudes, the highest phenol content was recorded in hill honey (52.85 ± 3.47 mg GAE/100 g; Figure 1), while the lowest was recorded in mountain honey (40.42 ± 1.11 mg GAE/100 g; Figure 1). The hill honeys showed significantly higher flavonoid contents (5.21 ± 0.55 mg QUE/100 g; p < 0.001), followed by mountain honeys (4.16 ± 0.55 mg QUE/100 g honey), with plain honeys having the lowest content (3.66 ± 0.4 mg QUE/100 g honey).

The biological activities and nutraceutical properties of honey are also linked to the presence of phenolic substances that play an important role [34,35]. In fact, the antioxidant action of phenolic acids is closely linked to their chemical structure and manifests itself through different mechanisms [36]. The main responsible factors are hydroxyl groups and their substitutions in the aromatic rings [37] that allow for free radicals to be swept away by giving up a hydrogen atom, electrons or metal cations. As reported by Gutiérrez-Grijalva et al. [38], the nutraceutical and biological properties are also driven by the binding of these compounds to organic acids and sugars that preserve the structure of the phenols themselves. Oxidative reactions involve numerous substances and are extremely complex; therefore, in order to evaluate the antioxidant capacity of honey, it is recommended to use at least two tests to increase the reliability of the experiment [39,40]. In our study, antioxidant activity was evaluated using ABTS spectrophotometric tests that evaluate the activity of both hydrophilic and lipophilic antioxidants, while the FRAP test uses antioxidants as reducing agents in a redox-linked colorimetric method in order to reduce the Fe³⁺/Fe²⁺ couple [40]. In general, variability in the antioxidant capacity values was found among the honeys studied (Figure 1). In particular, the highest FRAP antioxidant activity was observed in mountain and hill honeys at 2.75 and 2.67 ± 0.1 μmol TE/g, respectively, while the lowest activity was observed in the plain honey samples at 1.61 ± 0.06 μmol TE/g (p < 0.001). The same trend was observed for the ABTS values, which were significantly lower in the honeys produced in the plains (2.12 ± 0.08 μmol TE/g; p < 0.001), while the mountain honeys had the highest value (6.12 ± 0.22 μmol TE/g) although not statistically different from those of the hills (5.94 ± 0.19 μmol TE/g; p > 0.05). These results are in line with what was observed by Neupane et al. [41] in Nepalese honeys. The observed variations in the contents of phenolic components were predictable since the amounts of phytochemicals, such as phenolic acids and flavonoids, in honey are strictly related to the botanical origin, as highlighted by numerous investigations. Furthermore, plant metabolism changes according to the agro-climatic zone, influencing the composition of the nectar that bees feed on for honey production [42]. Furthermore, Mouhoubi-Tafinine et al. [43] highlighted that the antioxidant capacity of honey is also influenced by non-phenolic compounds, such as vitamins and amino acids, but also by antioxidant enzymes such as glucose oxidase and catalase.

3.2. Mineral Contents in Multiflora Honeys

Honey is rich in minerals, and its composition is related to bee grazing [44] and is therefore significantly influenced by botanical taxa in the vicinity of the site [45] but also by the geographical area and soil composition [46,47]. The presence of various elements in honey is also influenced by anthropogenic factors, such as beekeeping practices, environmental pollution and honey processing [2,48].

Table 1. Concentrations of elements (mg kg⁻¹ and μg kg⁻¹) in multiflora honey collected in different altimetric zones (plain, hill and mountain areas).

Elements	Plain		Hill		Mountain
	µ	SD	µ	SD	µ	SD
mg kg−1
Ca	4.44 b	0.54	5.39 a	0.66	4.87 ab	0.47
Mg	13.73 c	1.51	20.74 b	1.83	23.34 a	1.86
Na	52.48 a	5.42	52.60 a	6.33	36.25 b	4.57
µg kg−1
Ag	0.37 a	0.04	0.35 a	0.05	0.37 a	0.04
Al	1384.81 b	155.16	1217.43 b	118.91	1673.32 a	238.04
As	0.91 a	0.15	0.83 a	0.14	0.87 a	0.16
Ba	171.38 a	19.3	134.49 b	23.59	146.35 b	24.34
Cd	0.91 a	0.12	0.38 b	0.07	0.40 b	0.05
Co	4.58 a	0.66	3.32 b	0.74	3.40 b	0.58
Cr	16.77 a	1.81	15.68 a	2.15	12.63 b	1.24
Cu	248.21 a	28.98	267.90 a	47.95	280.53 a	55.27
Fe	1615.14 a	239.69	617.31 b	94.15	1637.43 a	301.41
Li	10.78 a	0.94	7.97 b	0.85	8.91 b	1.5
Mn	254.89 c	30.18	318.72 b	45.63	454.42 a	83.7
Mo	4.25 b	0.46	5.31 a	0.88	4.52 ab	0.96
Ni	34.58 a	4.47	26.35 b	4.60	33.90 a	2.46
Pb	7.51 b	0.72	8.38 b	1.26	12.92 a	1.64
Sn	29.07 a	3.16	32.45 a	5.05	29.86 a	3.53
Sr	124.27 a	14.34	79.71 b	10.62	120.06 a	15.24
Ti	133.38 ab	16.03	118.75 b	18.53	146.86 a	23.99
V	2.33 a	0.21	1.67 b	0.28	1.85 b	0.31
Zn	975.27 a	114.49	971.84 a	157.34	990.28 a	147.51

^a,b,c Means within a row with different superscripts differ p < 0.05.

reports a composition of trace elements, macro-elements and heavy metals in honey samples from the Basilicata region classified as plain, hill and mountain honeys. Trace elements Be, Hg, Sb and Se were not included in the statistical analysis since in all of the samples, a concentration lower than the LOQs was found. The LOQs for Be, Hg, Sb and Se are 0.12, 0.56, 3.5 and 3.8 µg/kg, respectively. A statistical analysis revealed significant differences between honeys from different altimetric zones, except for the Zn and Ag contents (p > 0.05), which could be related to production and conservation practices common among companies from different collection zones [49]. In addition, there is a low silver content in soil [50]. Significant differences were found between plain and hill honeys for all the elements studied, except for the Na, Cu, Pb and Ti contents; no significant differences were found between plain and mountain honeys for Fe, Ni and Mo contents. Furthermore, no significant differences were found between hill and mountain honeys for the As, Co, Cu, Sn, Sr, Ba and Cd contents.

When comparing the altitude zones, it was observed that mountain honeys had a significantly lower Na content (p < 0.05), while the Mg content was higher (p < 0.05) than that pf honeys from other zones (Table 1). Furthermore, these samples showed a higher Pb content (12.92 µg/kg, p < 0.05) than honeys from other zones, while the Mn content was lower in honeys from the plain zone than other zones (254.89 µg/kg, p < 0.05). Plain honeys had higher contents of Cr (16.77 µg/kg), V (2.33 µg/kg, p < 0.05), Ni (34.58 µg/kg), Co (4.58 µg/kg, p < 0.05), Sr (124.27 µg/kg), Ba (171.38 µg/kg, p < 0.05) and Cd (0.91 µg/kg, p < 0.05).

The richness of some macro- (Ca, K, Mg and Na) and microminerals (Co, Cr, Cu, Fe, Mn, Ni, Se and Zn) has important functions in various biochemical processes as constituents of many bioactive compounds [51]. The presence and content of minerals in honey reflect the area surrounding the apiary; bee foraging activity extends for about 10 km², and when bees collect nectar or pollen, these elements are transferred, contributing to the levels in honey. Pb, Cd and As do not perform any biological function and are highly toxic elements [52], and their presence in honey indicates environmental pollution [3]. In particular, the Pb content was found to be lower than that of Mediterranean honeys [53], and above all, it was not at the maximum limit (0.1 mg/kg) established by EC Regulation 2015/1005. The average Cd content in the studied honeys was 0.56 µg kg⁻¹, lower than that found in honeys collected in different Italian areas [54,55,56], and lower than that reported for honeys collected in six continents [2]. The average arsenic content found was 0.85 µg kg⁻¹, a value considerably lower than that found in Italian multifloral honeys [47].

3.3. Correlation

In this study, the correlation between the analyzed parameters of the different honey samples was evaluated, and the results are reported in Figure 2. Many authors reported that the phenolic component is the first factor influencing the antioxidant properties of honey; indeed, a significant correlation coefficient was shown between antioxidant activity and the total phenolic content [57,58]. In our study, however, the Pearson correlation coefficients (r) showed a low, if not almost null, relationship between total phenolics and FRAP (r = 0.014) and ABTS (r = 0.094) antioxidant activity. On the contrary, the r value was significantly higher between the total flavonoid content and FRAP (r = 0.37, p < 0.01) and ABTS (r = 0.565; p < 0.001) in line with the results reported in our previous work [54]. Our results show that in the studied honeys, the antioxidant capacity does not depend only on phenolic compounds but would be influenced by other factors such as organic acids, amino acids, proteins and ascorbic acid, which act in synergy with the phenolic compounds of honey. Similar results were found by Becerril-Sánchez et al. [12], Shamsudin et al. [59], Khiati [60] and Idris et al. [61]. On the contrary, many authors reported a high correlation between the TPC and TFC with the antioxidant capacity of honeys [62,63]. A study of Argentine honeys showed a higher correlation of antioxidant activity with the TPC (r = 0.91) than with the flavonoid content (r = 0.51) [30]; differently, in Malaysian honey, a higher correlation was recorded with the TFC (r = 0.408) than with the TPC (r = 0.385) [59]. Al-Farsi et al. [64] observed a negative correlation between phenols and a DPPH assay (IC50) (r = −0.616) in Omani honey and in some European honeys (TPC, r = −0.319; TFC, r = −0.386) [65].

The low correlations we found indicate that the antioxidant activity is not only promoted by polyphenols but also by other biologically active substances (catalase, ascorbic acid and proteins) which contribute to the antioxidant activity of honey, as reported by Muflihah et al. [66].

Figure 2 shows the correlation between the metal content and polyphenol content and antioxidant activity. A statistically significant correlation (p < 0.001) was observed between total phenols and Na (r = 0.51), Ca (r = 0.35), Cu (r = 0.31) and Sn (r = 0.31), while the correlation coefficients had negative correlations with the contents of Al, Fe, Ni, Pb, Sn, Li and Ti (r = −0.63, −0.71, −0.48, −0.61, −0.41 and −0.41, respectively; p < 0.01). The flavonoid content was positively correlated with Mg and Ca (r = 0.41 and 0.53, respectively; p < 0.001), while a higher negative correlation was observed with the Fe, V, Ni, Co, Sr, Cd, Li and Ti contents (r = −0.84, −0.62, −0.69, −0.52, −0.72, −0.60, −0.65 and 0.44, respectively; p < 0.001). Our results also show a positive and statistically significant correlation (p < 0.001) between the metal content and antioxidant activity. In particular, high r values and high statistical significance (p < 0.001) were observed in the correlations between the Mg, Ca, Mn, Cu, Mo and Pb contents and FRAP and ABTS values. Strong significant but negative correlations were found between Cd, Co, Fe and Cr contents and FRAP and ABTS values. Pohl et al. [67] observed how some minerals, including Cd, Fe and Mn, interact with numerous organic components (organic acids, proteins, amino acids, polyphenols, vitamins and aromatic compounds), forming stable complexes in honey. Moridani et al. [68] instead hypothesized that thanks to the ability of metals to act as free radical acceptors, they are able to enhance the biological activity of an organic component. Chaoui et al. [69] demonstrated how some antioxidant enzymes are activated in response to oxidative stress from Cd and Zn in Phaseolus vulgaris L.

3.4. Correlation Between Altitude and Studied Parameters

In Figure 2, correlation matrices showing the relationships between altitude and total phenols and total flavonoids are reported. The correlation between altitude and TPC was negative, intermediate and not significant (r = −0.30), while with flavonoids, it was positive and intermediate but not significant (r = 0.20). These results suggest that altitude is only partly responsible for the TPC and TFC of multifloral honey samples. The TPC and TFC are phytochemicals in plants whose biosynthesis is influenced by several environmental factors, such as the average temperature, soil, atmospheric pressure, duration of vegetation period and radiation intensity, which in turn are influenced by the cultivation altitude [70].

Suleiman et al. [13] observed a higher phenolic content in Ziziphus honey samples collected at low altitudes due to higher nectar secretion due to more favorable ambient temperature.

In this study, high and significant correlations were found between altitude and antioxidant activity using FRAP and ABTS (p < 0.001; Figure 2). Shakoori et al. [71] reported that the increase in antioxidant activity in honey depends on the abundance of some plant essences that characterize the bee pasture as well as the botanical origins; furthermore, the presence of these essences is strongly influenced by the altitude that determines ecological and geographical changes. In support, Teron et al. [72] highlighted the importance of altitude in influencing the chemical characteristics of plant nectar that determine changes in metabolites in honey, especially the components that promote health. Health benefits are influenced by the bioavailability of phytochemicals and their absorption and the metabolic pathways detected in honey [73]. In general, it is known that polyphenols are responsible for the activity of the intestinal microbiota and its composition, and they are involved in phase I of metabolism (oxidation, reduction, hydrolysis, etc.) and phase II (conjugation) [74].

Positive and significant correlations (p < 0.001) were observed between altitude and Mg (r = 0.87), Al (r = 0.51), Mn (r = 0.83) and Pb (r = 0.85), while negative correlations were observed between altitude and Na (r = −0.74), Cr (r = −0.71), V (r = −0.47), Co (r = −0.53) and Cd (r = −0.76), and therefore, as the altitude increases, the presence of these minerals decreases. Minerals detected in honey come from both natural sources (soil and plants) and anthropogenic sources [3]. Tew et al. [75] highlighted the effect of soil type on plant nectar production. Depending on the minerals present in the soil, there is a positive effect on the production of flowers and, consequently, on the quantity of the nectar of plants visited by bees. For example, Cardoso et al. [76] showed that Mn influences the number of flowers of plants and the quantity of the total nectar; again, the positive influence of the mineral composition of the soil on the production of nectar was confirmed for another milleritic plant, Allium ursinum L. Schmidlová et al. [45] highlighted the correlation between the contents of some elements, such as Fe, Mn and Ca, and the type of soil.

In support, Loix et al. [77] and Schmidlová et al. [45] reported that the mineral profile of honey can be influenced by the type of soil on which the hive is located and the soil on which the pasture is located; however, the soil and pasture are influenced by altitude. In conclusion, depending on the altitude, honey can be more or less rich in mineral components important for the health of the consumer, such as Zn, Co, Mn and others which are implicated in numerous biochemical processes.

4. Conclusions

The effect of altitude on multifloral honeys was highlighted on all of the parameters analyzed. Our results show that honeys produced in the hills had higher contents of both total phenols and flavonoids, and the antioxidant activity was higher in mountain honeys. The mineral profile was extremely variable in the three study areas. Furthermore, there was a negative correlation between total phenols and altitude, while there was a positive but low correlation with TFC, suggesting that altitude was only partly responsible for the TPC and TFC.