Tryptophan and Biogenic Amines in the Differentiation and Quality of Honey.

Honey is a natural product with beneficial properties to health and has different characteristics depending on the region of production and collection, flowering, and climate. The presence of precursor amino acids of- and biogenic amines can be important in metabolomic studies of differentiation and quality of honey. We analyzed 65 honeys from 11 distinct regions of the State of Santa Catarina (Brazil) as to the profile of amino acids and biogenic amines by HPLC. The highest L-tryptophan (Trp), 5-hydroxytryptophan (5-OH-Trp), and tryptamine (Tryp) levels were detected in Cfb climate and harvested in 2019. Although we have found high content of serotonin, dopamine, and L-dopa in Cfb climate, the highest values occurred in honey produced during the summer 2018 and at altitudes above 900 m. Results indicate that the amino acids and biogenic amine levels in honeys are good indicators of origin. These data warrant further investigation on the honey as source of amino acids precursor of serotonin, melatonin, and dopamine, what can guide the choice of food as source of neurotransmitters.

Introduction

Honey is known for its antimicrobial, anti-inflammatory, anticancer, antiatherogenic, antithrombotic, and antioxidant effects, in addition to its analgesic activity in the human body. Conscious consumption and the search for foods with good nutritional quality, aiming at the prevention of diseases, has been a priority in the human diet. In addition, there is also an interest in eating foods that provide well-being, both physical and emotional. Phenolic compounds seem to be the most important constituents of honey, responsible for its antioxidant activity. However, bioactive amines, as well as their precursor amino acids, have been the subject of many studies, due to the great interest regarding the nutritional paradox and their possible action as an antioxidant/well-being agent. These substances are also related to regulation of the cell cycle and play a fundamental role in the synthesis route of important neurotransmitters, such as serotonin and dopamine.

Some studies report the presence of biogenic amines in samples of honey and other bee-derived products (ie, propolis), which have biological properties and can be used beneficially in the food and pharmaceutical industry. The presence of free amino acids in bee products can lead to the formation of biogenic amines (BA), which may be desirable or undesirable in food products, making further research essential.

The ingestion of L-tryptophan (L-Trp) and 5-hydroxytryptophan (5-OH-Trp), for example, is essential for the formation of serotonin in the brain. Serotonin, a neurotransmitter, does not cross the blood–brain barrier and the synthesis and turnover of this monoamine depend on the intake of amino acids. In humans, due to its essentiality, the recommended daily dose of L-Trp is around 4 mg/kg body weight per day for adults and 12 mg/kg body weight for children. Thus, foods containing higher levels of L-Trp and 5-OH-Trp can help balance serotonin levels. It is noteworthy that honey is a source not only of L-Trp, but also of other metabolites derived from this amino acid and important to human health, such as nicotinamide (vitamin B6), melatonin, tryptamine, kynurenine, 3-hydroxykynurenine, and quinolinic and xanthurenic acids. Furthermore, among other parameters, amino acid content has also been proposed as a method to determine the botanical and/or geographic provenance of food products, and honey has often been the target of this approach.

Honey and its derivatives have been increasingly valued natural substances; however, their physicochemical and biological properties are determined by many factors, including bee species, the nectar donor plant (specific for each season), geographic area/origin, seasonal conditions, harvesting and storage processes, and climatic conditions. Therefore, it is important to expand the information on chemical composition and explore the biological activity of honeys from different geographic regions and methods of extraction, among other things. Due to its high complexity, the chemical analysis of honey poses a considerable challenge. Therefore, this study aimed to identify compounds from the biogenic amine class, as well as their precursor amino acids (ie, Trp and 5-OH-Trp) in honeys from different agro-ecological regions.

Methods

Honey collection places

Samples of honey (65) harvested in 2018 to 2019 were kindly provided by beekeepers from 11 agroecological regions of Santa Catarina State (Brazil), namely: (1) Planalto Serrano de São Joaquim, (2) coast, Itajaí, and Tijucas River Valleys, (3) coast of Florianópolis and Laguna, (4) Alto Vale do Itajaí, (5) Carboniferous, Extreme South, and Colonial Serrana, (6) Uruguai River Valley, (7) Alto Vale do Rio do Peixe and Alto Irani, (8) Central Plateau, (9) Northern Santa Catarina Plateau, (10) Santa Catarina Northwest, and (11) Campos de Lages (Table 1). All honey samples are polyfloral, because, in addition to presenting the predominant flowering described by beekeepers, they all contain pollen from native species of the regions of origin.

Table 1.

Location and predominant flowering of honey from Santa Catarina (flowering year/collection and season).

Sample	Region	City	Altitude (m)	Climate (Koppen-Geiger)	Predominant flowering	Year of collection	Season
1A1	Coast, Itajaí, and Tijucas river valleys	Benedito Novo	683	Humid subtropical climate (Cfa)	Cinnamon and Piptocarpha angustifolia	2019	Summer
1A2		Itapoá	457		Wild flowers	2018	Spring
1A3		Itapoá			Wild flowers	2019	Summer
1A4		Nova Trento	156		Wild flowers	2018	Spring
1A5		Nova Trento			Eucalyptus	2019	Autumn
1B1		Jaguaruna	92		Eucalyptus	2019	Autumn
1B2	Coast of Florianópolis and Laguna	Balneário Gaivota	9	Humid subtropical climate (Cfa)	Wild flowers	2019	Spring
1B3		Jaguaruna	92		Wild flowers	2018	Autumn
1B4		Jaguaruna			Eucalyptus	2019	Spring
1B5		Major Gercino	42		Wild flowers	2018	Autumn
1B6		Major Gercino			Eucalyptus	2019	Autumn
2A1	Alto Vale do Itajaí	José Boiteux	726	Humid subtropical climate (Cfa)	Baccharis dracunculifolia	2018	Spring
2A2		José Boiteux	491		Dracena frangans and Holvenia dulcis	2018	Spring
2A3		José Boiteux	717		Eucalyptus	2019	Autumn
2A4		Salete	593		Holvenia dulcis	2018	Spring
2A5		Vidal Ramos	763		Baccharis dracunculifolia	2019	Summer
2A6		Vidal Ramos	493		Holvenia dulcis	2019	Summer
2A7		Vidal Ramos	761		Baccharis dracunculifolia	2019	Summer
2B1	Carboníferous, Extreme South, and Colonial Serrana	Anitápolis	760	Humid subtropical climate (Cfa)	Baccharis dracunculifolia, Piptocarpha angustifolia, trimera and Holvenia dulcis	2018	Spring
2B2		Anitápolis			Eugenia sp., Holvenia dulcis, Clethra scabra, and Dracena fragans	2019	Summer
2B3		Orleans	600		Wild flowers	2018	Spring
2C1	Uruguai River Valley	Descanso	272	Humid subtropical climate (Cfa)	Holvenia dulcis	2018	Spring
2C2		Descanso			Eucalyptus	2019	Autumn
2C3		Itapiranga	300		Eucalyptus	2019	Autumn
2C4		Itapiranga			Holvenia dulcis	2019
2C5		Saudades	364		Holvenia dulcis	2018	Spring
2C6		Saudades			Eucalyptus	2019	Autumn
3A1	Alto Vale do Rio do Peixe and Central Plateau	Curitibanos	995	Humid temperate climate (Cfb)	Wild flowers	2019	Summer
3A2		Curitibanos			Wild flowers	2019	Summer
3A3		Erval Velho	699		Native fruit tree	2018	Spring
3A4		Erval Velho			Holvenia dulcis	2018	Spring
3A5		Erval Velho			Baccharis dracunculifolia and Holvenia dulcis	2019	Summer
3A6		Luzerna	725		Cinnamomum verum	2018	Spring
3A7		Luzerna			Holvenia dulcis	2018	Spring
3A8		Luzerna			Wild flowers	2019	Summer
3B2	Northern Santa Catarina Plateau	Bela Vista do Toldo	793	Humid temperate climate (Cfb)	Sebastiania commersoniana and Campomanesia xanthocarpa	2018	Spring
3B3		Três Barras	766		Sebastiania commersoniana	2018	Spring
3B5		Rio Negrinho	982		Wild flowers	2018	Spring
3B6		Rio Negrinho	982		Wild flowers	2019	Summer
3B7		Santa Terezinha	625		Holvenia dulcis	2018	Spring
3C1	Santa Catarina Northwest	Campo Erê	894	Humid temperate climate (Cfb)	Holvenia dulcis	2019	Summer
3C2		Campo Erê	650		Eucalyptus	2019	Autumn
3C3		Dionísio Cerqueira	856		Holvenia dulcis	2018	Spring
3C4		Dionísio Cerqueira			Eucalyptus	2019	Autumn
3C5		Xaxim	598		Holvenia dulcis	2018	Spring
4A1	Lages Field	Bocaina do Sul	980	Humid temperate climate (Cfb)	Campomanesia xanthocarpa, Piptocarpha angustifolia, Eugenia sp., and Vochysia tucanorum	2018	Spring
4A2		Bocaina do Sul			Clethra scabra	2019	Summer
4A3		Bom Retiro	522		Holvenia dulcis	2019	Summer
4A4		Bom Retiro	944		Clethra scabra	2019	Summer
4A5		Capão Alto	1007		Native plants	2018	Spring
4A6		Capão Alto			Wild flowers	2019	Summer
4B1	Alto Vale do Rio do Peixe and Alto Irani	Água Doce	1203	Humid temperate climate (Cfb)	Piptocarpha tomentosa	2018	Spring
4B2		Água Doce	1203		Ocotea porosa	2018	Spring
4B3		Água Doce	1203		Baccharis trimera	2019	Summer
4B4		Lebon Régis	1280		Cinnamon, Piptocarpha angustifolia, Clethra scabra, Baccharis uncinella, and Cupania vernalis	2018	Spring
4B5		Lebon Régis	1280		Vernonia polysphaera and Baccharis trimera	2019	Autumn
4B6		Matos Costa	973		Astronium fraxinifolium, Lithrea brasiliensis, and Ilex theezans	2018	Spring
4B7		Matos Costa	1156		Clethra scabra	2019	Autumn
5(1)	Planalto Serrano de São Joaquim	Bom Jardim da Serra	1272	Humid temperate climate (Cfb)	Piptocarpha angustifolia, Lithraea molleoides, Astronium fraxinifolium, Baccharis trimera, and Brosimum gaudichaudii	2018	Spring
5(2)		Bom Jardim da Serra	1247		Senna bicapsularis, Struthanthus flexicaulis, and Baccharis trimera	2019	Autumn
5(3)		São Joaquim	1276		Piptocarpha angustifolia, Lithrea brasiliiensis, Astronium fraxinifolium, and Baccharis trimera	2018	Spring
5(4)		São Joaquim	1309		Eupatorium sp., Struthanthus flexicaulis, Baccharis trimera, and Strychnos pseudoquina	2019	Autumn
5(5)		São Joaquim	1148		Piptocarpha angustifolia and Mimosa scabrella	2018	Spring
5(6)		São Joaquim	1051		Senna bicapsularis	2019	Summer
5(7)		São Joaquim	1055		Wild flowers	2019	Summer

Extraction and chromatographic analysis

Biogenic amines (serotonin—Ser, melatonin—Mel, dopamine—Dop, and tryptamine—Tryp), L-Trp, 5-OH-Trp, and L-dopa were extracted as described by Lima et al. Honey samples (1 g) were mixed with 3 mL of perchloric acid (5%, v/v), homogenized (vortex, 1 minute) and incubated in a cold ultrasonic bath (30 minutes), followed by centrifugation (10 minutes, 6000g, 5°C). The supernatant containing the free amines and amino acids was collected and subjected to derivatization using supernatant, 4.5 mol/L Na2CO3 and 18.5 mmol/L dansyl-chloride in acetone. The solution was kept at 60°C for 1 hour and, in sequence, proline (1 mg/mL) was added and the mixture kept in the dark for 1 hour and homogenized by vortexing every 15 minutes. After this interval, toluene (HPLC grade) was added, the mixture was vortexed (1 minute) and the supernatant (hydrophobic part containing the target compounds) was dried in a nitrogen line.

The samples were resuspended in acetonitrile (HPLC grade), homogenized by vortexing and incubated (1 minute) in an ultrasonic bath, followed by filtration (0.25 μm) and injection in a high-performance liquid chromatograph (HPLC; Dionex UltiMate 3000; Thermo Fisher Scientific, Bremen, Germany), according to Dadáková et al with modifications. Briefly, 20 μL of sample was injected into an ACE C18 reverse phase column (4.6 × 250 mm; 5 µm), thermostatted at 25°C, coupled to a quaternary automatic sampler 3000 pump and diode array detector (DAD-3000RS). Amines and amino acids were eluted in a gradient system, as follows: 0 to 2 minutes, 40% A + 60% B; 2 to 4 minutes, 60% A + 40% B; 4 to 8 minutes, 65% A + 35% B; 8 to 12 minutes, 85% A + 15% B; 12 to 15 minutes, 95% A + 5% B; 15 to 21 minutes, 85% A + 15% B; 21 to 22 minutes, 75% A + 25% B; 22 to 25 minutes, 40% A + 60% B. Identification of biogenic amine and amino acids of interest (eg, L-Trp, 5-OH-Trp, Tryp, L-dopa, Ser, Mel, and Dop) was based on the retention times of analytical standards (Sigma-Aldrich, MO, USA), with detection at λ = 225 nm. For the purposes of compound quantification (µg/100 g), the peak areas were integrated using Chromeleon 7 software (Thermo Fisher Scientific, Bremen, Germany). The limit of detection (LOD), limit of quantification (LOQ), linear regression, regression coefficient (R2), recovery, and repeatability values (n = 6) observed are shown in Table 2. Repeatability (below 5%) was determined using 6 replicates/honey samples chosen at random. Mean recovery (%, n = 6) was tested with 5 concentrations (12.5, 50, 100, 150, and 200 mg/L) of analytical standard (Table 2).

Table 2.

LOD, LOQ, linear regression calibration curves, regression coefficient (R2), recovery, and repeatability of biogenic amines, by the analytical method in samples of honey.

Aminoacids/biogenic amines	LOD a (mg/L)	LOQ (mg/L)	Regression equation	R2	Recovery b values (%)	Repeatability c
Tryptophan	0.019	0.104	y = 0.3614x + 0.1297	.99	96.5	3.3
5-OH-tryptophan	0.026	0.102	y = 0.2574x + 0.0561	.99	97.3	3.1
Tryptamine	0.049	0.106	y = 4.232x + 42.342	.99	94.8	4.1
Serotonin	0.013	0.085	y = 4.0423x + 19.911	.99	96.7	3.2
Melatonin	0.013	0.102	y = 1.3749x + 0.9985	.99	92.3	3.9
L-dopa	0.018	0.110	y = 205.1x − 0.012	.99	91.9	3.5
Dopamine	0.029	0.099	y = 3.822x + 71.767	.98	101.2	4.1

Range for amino acids and biogenic amines 12.5 to 200 mg/L.

n = 6.

n = 6.

Statistical analysis

Data on biogenic amines and their precursors found in honey samples were submitted to analysis of variance (ANOVA), and if the data were significant, they were submitted to the Scott Knott test (P < .05). The ANOVA and the mean comparison test were performed using SISVAR statistical software. Principal component analysis (PCA; XLSTAT software, version 2020; Addinsoft, France) was applied to visualize the possible correlation between amino acid content and the different classes of biogenic amines and the agroecological regions where the samples were collected.

Results and discussion

Currently, honey and its products have been valued as natural foods. However, it and its physicochemical and biological properties are affected by several factors of (a)biotic nature. The results clearly reveal that the composition of floral honeys produced in southern Brazil is dependent on several factors, including the geographical area of production, that is, the agroecological region of production and collection, as well as the nectar donor plants (specific for each season), as also verified in other similar studies.

In an attempt to establish a descriptive model for the grouping of precursor amines and amino acids according to the different agroecological regions of collection, as well as flowers from which nectar is collected, it was decided to compare the results obtained by PCA. Honeys from the coast of Florianópolis and Laguna (1B2) (Cfa climate, humid subtropical climate – harvested in spring 2019), from the Northern Plateau of Santa Catarina (3B2, harvested in spring 2018), and from the agroecological region of the Planalto Serrano de São Joaquim (Cfb climate – temperate oceanic climate) (5.5 and 5.7, harvested in summer 2018 and 2019) stood out for their L-Trp content (PC1+ and PC2+). Honeys from Cfa climate (Alto Vale do Itajaí—2A6 and Uruguai River Valley—2C3) harvested in 2019 (summer and autumn, respectively) are distinguished from the others by having the highest tryptamine content (35.01 and 35.43 µg/100 g, respectively). Both L-Trp and Tryp are precursors of Ser and their presence may be important for the control of some physiological disorders, such as obsessive-compulsive disorder. Ser and Dop (r = .89, P < .05), as well as the amino acid L-dopa with these amines, showed a significant and strong correlation (Ser: r = .76 and Dop: .79, P < .05). Honey from the agroecological regions Alto Vale do Rio do Peixe and Alto Irani (4B6), harvested in Spring 2018, showed the highest levels of these amines (Ser: 495.15 µg/100 g; Dop: 33.98 µg/100 g) and the amino acid L-dopa (0.72 µg/100 g) (PC1+ and PC2−) (Figures 1 and 2B; Table 3).

Figure 1.

Two-dimensional projection and scores of profile of precursor amino acids and biogenic amines of floral honeys from the agroecological regions of the State of Santa Catarina. Honey samples are represented by blue points (see Table 1) and amino acids and biogenic amines by red points.

Abbreviations: 5-OH-TRP, 5-hydroxy-tryptophan; L-DOPA, DOP, dopamine; L-TRP, L-tryptophan; MEL, melatonin; SER, serotonin; TRYP, tryptamine.

Figure 2.

Two-dimensional projection and scores of profile of precursor amino acids and biogenic amines of floral honeys from the agroecological regions of the State of Santa Catarina analyzed as to the time of harvest (A) and season (B). Honey samples are represented by blue points (see Table 1) and amino acids and biogenic amines by red points.

Abbreviations: 5-OH-TRP, 5-hydroxy-tryptophan; L-DOPA, DOP, dopamine; L-TRP, L-tryptophan; MEL, melatonin; SER, serotonin; TRYP, tryptamine.

Table 3.

Precursor amino acids and biogenic amines (µg/100 g) of floral honeys from the agroecological regions of the State of Santa Catarina (harvest 2018 and 2019).

Honey	L-Trp 1	5-OH-Trp 2	Tryp 3	Ser 4	Mel 5	L-DOPA 6	Dop 7
1A1	749.08 ± 33.86p	1314.04 ± 1.36d	15.58 ± 2.47g	21.27 ± 1.93l	25.64 ± 3.23h	0.07 ± 0.04h	12.79 ± 0.60b
1A2	930.66 ± 7.18o	nd	22.47 ± 0.20d	55.63 ± 0.79c	nd	0.02 ± 0.00j	1.68 ± 0.22j
1A3	875.64 ± 0.45o	nd	12.36 ± 0.14h	20.68 ± 0.51l	nd	nd	2.62 ± 0.17i
1A4	970.84 ± 9.24o	nd	17.21 ± 0.19f	24.11 ± 0.52k	11.99 ± 0.74k	0.03 ± 0.00j	2.32 ± 0.09i
1A5	1612.14 ± 46.25j	989.14 ± 67.77g	15.26 ± 1.29g	26.32 ± 1.75i	24.54 ± 5.72i	0.05 ± 0.00h	3.93 ± 0.02g
1B1	1408.79 ± 19.32k	708.82 ± 3.43j	12.60 ± 0.18h	23.80 ± 0.14k	nd	0.02 ± 0.00j	1.48 ± 0.05j
1B2	3358.65 ± 13.70a	764.09 ± 0.06i	18.24 ± 1.93f	29.10 ± 0.25h	nd	0.04 ± 0.00i	1.66 ± 0.07j
1B3	387.66 ± 23.42r	1188.36 ± 53.31e	10.63 ± 0.18i	10.21 ± 0.16r	36.16 ± 8.71g	0.02 ± 0.00j	4.08 ± 0.14g
1B4	1113.56 ± 36.04n	864.93 ± 78.17h	13.83 ± 1.94h	18.83 ± 0.73m	93.26 ± 12.95a	0.09 ± 0.04g	3.86 ± 1.03g
1B5	1150.29 ± 19.44n	36.76 ± 0.86t	8.25 ± 0.07j	15.26 ± 0.23o	20.85 ± 3.97i	0.02 ± 0.01j	2.99 ± 0.21h
1B6	1224.87 ± 13.32m	749.16 ± 14.09i	10.88 ± 2.20i	12.01 ± 0.02q	nd	0.01 ± 0.00j	1.96 ± 0.18j
2A1	1226.04 ± 11.92m	112.67 ± 0.06s	11.44 ± 0.00i	13.16 ± 0.21p	nd	0.05 ± 0.00h	1.55 ± 0.00j
2A2	1316.26 ± 142.72l	79.55 ± 30.53s	18.00 ± 0.12f	2.75 ± 0.10v	nd	0.09 ± 0.00g	1.12 ± 0.01k
2A3	1081.90 ± 86.28n	1210.13 ± 90.22e	22.51 ± 0.39d	17.73 ± 0.06n	34.23 ± 1.30g	0.03 ± 0.02i	1.10 ± 0.13k
2A4	263.31 ± 21.91s	22.99 ± 0.44t	6.24 ± 0.07k	7.62 ± 0.21s	nd	nd	Nd
2A5	1032.62 ± 14.80o	90.07 ± 32.13s	21.26 ± 0.22d	6.76 ± 0.12t	nd	0.02 ± 0.00j	1.60 ± 0.17j
2A6	1083.81 ± 35.33n	494.50 ± 102.30m	35.01 ± 0.92a	4.74 ± 0.07u	30.19 ± 4.83h	0.03 ± 0.00j	1.01 ± 0.08k
2A7	944.98 ± 21.23o	34.40 ± 0.82t	11.90 ± 0.06i	13.50 ± 0.05p	19.17 ± 0.63i	0.02 ± 0.00j	1.73 ± 0.09j
2B1	1504.82 ± 4.88k	98.67 ± 13.70s	23.57 ± 0.20c	7.80 ± 0.40s	nd	0.07 ± 0.00h	1.03 ± 0.01k
2B2	576.57 ± 6.24q	48.96 ± 5.73t	20.51 ± 0.10e	2.22 ± 0.15v	nd	0.03 ± 0.01j	0.63 ± 0.07k
2B3	627.68 ± 7.54q	57.28 ± 11.45t	14.58 ± 0.02g	26.92 ± 0.75i	21.19 ± 2.64i	0.01 ± 0.00k	1.90 ± 0.18j
2C1	602.91 ± 6.76q	337.50 ± 81.44o	16.95 ± 0.94f	12.58 ± 0.79p	16.33 ± 1.14j	nd	0.84 ± 0.06k
2C2	1191.99 ± 28.47m	141.90 ± 27.21r	14.47 ± 2.67g	8.02 ± 0.07s	12.21 ± 2.42k	0.01 ± 0.00j	1.75 ± 0.06j
2C3	836.37 ± 11.59p	182.38 ± 80.60r	35.43 ± 0.81a	7.16 ± 0.57t	nd	0.10 ± 0.03g	1.32 ± 0.13j
2C4	377.62 ± 5.99r	111.11 ± 3.78s	12.35 ± 0.15h	5.43 ± 0.07u	14.15 ± 1.56k	nd	0.71 ± 0.22k
2C5	743.89 ± 32.08p	138.26 ± 3.26r	26.97 ± 0.41b	5.94 ± 1.48t	17.43 ± 0.67j	nd	1.13 ± 0.02k
2C6	1764.97 ± 20.53i	1156.40 ± 2.59f	17.62 ± 1.83f	8.24 ± 0.30s	20.01 ± 0.72i	0.01 ± 0.00j	1.44 ± 0.30j
3A1	1666.18 ± 15.98j	343.50 ± 23.86o	11.77 ± 0.15i	15.27 ± 0.99o	17.87 ± 1.27j	0.02 ± 0.01j	0.90 ± 0.02k
3A2	967.73 ± 12.19o	423.85 ± 14.23n	15.39 ± 0.87g	10.84 ± 0.36r	nd	0.06 ± 0.00h	1.23 ± 0.02j
3A3	1515.81 ± 16.03k	624.56 ± 21.23k	24.17 ± 1.99c	7.10 ± 0.61t	nd	0.07 ± 0.00h	0.86 ± 0.02k
3A4	1096.43 ± 47.62n	173.39 ± 5.96r	13.59 ± 0.33h	8.09 ± 0.05s	nd	0.07 ± 0.00h	3.20 ± 0.04h
3A5	1207.37 ± 14.26m	664.09 ± 4.33j	15.31 ± 0.05g	8.80 ± 0.04r	nd	0.08 ± 0.00g	2.34 ± 0.12i
3A6	1381.80 ± 1.09k	156.42 ± 9.71r	17.69 ± 0.96f	11.28 ± 0.03r	27.75 ± 2.76h	0.02 ± 0.00j	1.24 ± 0.01j
3A7	1440.32 ± 17.23k	255.07 ± 16.63q	18.28 ± 0.01f	17.31 ± 0.20n	37.79 ± 0.35f	0.02 ± 0.00j	1.48 ± 0.10j
3A8	1466.00 ± 4.52k	973.13 ± 34.25g	18.31 ± 0.47f	12.98 ± 0.47p	nd	0.05 ± 0.02h	4.86 ± 1.04f
3B2	3158.17 ± 32.94b	352.68 ± 9.56o	21.29 ± 0.26d	37.01 ± 0.09e	nd	0.05 ± 0.00h	1.54 ± 0.19j
3B3	928.68 ± 1.88o	173.53 ± 9.38r	10.30 ± 0.01i	13.74 ± 0.01p	14.93 ± 2.44k	0.02 ± 0.00j	0.69 ± 0.04k
3B5	1729.82 ± 16.02i	777.89 ± 2.44i	20.38 ± 0.02e	13.97 ± 0.18p	nd	0.06 ± 0.01h	9.84 ± 0.06c
3B6	1228.09 ± 12.22m	221.52 ± 15.86q	16.89 ± 0.15f	8.82 ± 0.01r	nd	0.02 ± 0.00j	2.92 ± 0.03h
3B7	1075.95 ± 14.30n	287.19 ± 2.56p	17.75 ± 0.16f	9.61 ± 0.50r	nd	0.02 ± 0.00j	1.10 ± 0.04k
3C1	934.12 ± 3.76o	567.29 ± 13.81l	21.67 ± 0.81d	4.97 ± 0.00u	nd	0.01 ± 0.00k	0.93 ± 0.00k
3C2	1309.26 ± 25.95l	1547.30 ± 55.99c	17.76 ± 0.99f	21.38 ± 0.13l	nd	0.02 ± 0.00j	2.39 ± 0.07i
3C3	1378.81 ± 13.15k	87.45 ± 9.45s	16.50 ± 2.51g	51.52 ± 1.57d	58.63 ± 0.31d	0.53 ± 0.01b	8.49 ± 1.48d
3C4	637.20 ± 8.88q	161.84 ± 3.21r	11.99 ± 5.05i	12.98 ± 0.35p	17.60 ± 0.45j	0.01 ± 0.00k	2.51 ± 0.17i
3C5	954.75 ± 2.01o	108.54 ± 0.48s	16.13 ± 1.05g	14.92 ± 0.41o	41.77 ± 0.88e	nd	2.36 ± 0.62i
4A1	2250.72 ± 2.55f	119.28 ± 0.34s	15.59 ± 0.63g	31.31 ± 1.24g	nd	0.04 ± 0.01i	3.33 ± 0.37h
4A2	944.05 ± 2.95o	109.71 ± 1.51s	27.22 ± 1.02b	11.40 ± 0.52r	nd	0.32 ± 0.00c	4.68 ± 0.07f
4A3	1451.82 ± 59.95k	155.58 ± 33.77r	14.61 ± 0.47g	10.56 ± 2.09r	44.49 ± 6.87e	0.06 ± 0.00h	3.96 ± 0.07g
4A4	1452.47 ± 51.42k	76.18 ± 7.58s	12.37 ± 0.78h	24.81 ± 0.74j	nd	0.03 ± 0.00i	1.54 ± 0.06j
4A5	826.83 ± 0.73p	71.95 ± 5.36s	10.85 ± 0.54i	24.71 ± 0.25j	nd	0.01 ± 0.00k	1.23 ± 0.01j
4A6	1291.53 ± 36.42l	287.23 ± 11.21p	17.77 ± 0.40f	37.70 ± 0.44e	nd	0.03 ± 0.00j	2.40 ± 0.12i
4B1	1455.32 ± 30.95k	98.47 ± 10.14s	17.07 ± 0.33f	33.04 ± 0.24f	22.05 ± 1.30i	0.00 ± 0.00k	0.00 ± 0.00l
4B2	2412.74 ± 59.28e	153.78 ± 2.29r	20.26 ± 0.41e	26.98 ± 0.54i	nd	0.09 ± 0.01g	2.48 ± 1.61i
4B3	2141.29 ± 10.93g	2045.59 ± 72.26a	18.65 ± 0.50f	12.05 ± 0.05q	45.15 ± 0.23e	0.09 ± 0.00g	Nd
4B4	1951.09 ± 17.79h	115.81 ± 7.38s	22.13 ± 1.20d	20.83 ± 1.09l	nd	0.05 ± 0.01h	0.68 ± 0.02k
4B5	837.81 ± 9.78p	76.73 ± 2.56s	16.03 ± 0.34g	11.82 ± 1.30r	nd	0.09 ± 0.02g	0.69 ± 0.36k
4B6	884.27 ± 25.97o	99.36 ± 29.06s	17.41 ± 0.86f	495.15 ± 0.14a	nd	0.72 ± 0.00a	33.98 ± 0.20a
4B7	786.62 ± 8.64p	71.21 ± 1.31s	13.62 ± 0.25h	33.38 ± 0.44f	nd	0.03 ± 0.00j	1.74 ± 0.07j
5.1	626.98 ± 7.19q	1714.61 ± 28.85b	14.83 ± 1.48g	33.11 ± 0.54f	30.03 ± 1.32h	0.05 ± 0.00h	2.69 ± 0.03i
5.2	662.25 ± 7.29q	1700.99 ± 10.37b	15.95 ± 0.02g	23.05 ± 0.24k	32.83 ± 0.22g	0.05 ± 0.00h	2.97 ± 0.10h
5.3	1122.22 ± 46.81n	80.16 ± 6.03s	19.63 ± 2.77e	34.02 ± 3.20f	88.08 ± 2.31b	0.02 ± 0.00j	4.02 ± 0.99g
5.4	2024.21 ± 31.36h	70.86 ± 0.20s	15.89 ± 0.20g	16.87 ± 1.19n	35.24 ± 3.00g	0.02 ± 0.01j	1.85 ± 0.01j
5.5	2569.65 ± 92.00d	87.26 ± 5.95s	25.13 ± 0.13c	64.13 ± 0.05b	44.29 ± 2.48e	0.16 ± 0.03e	5.02 ± 0.02f
5.6	750.03 ± 28.87p	115.33 ± 15.91s	16.01 ± 0.32g	25.57 ± 1.65j	66.25 ± 3.76c	0.02 ± 0.00j	3.67 ± 0.65g
5.7	2827.54 ± 41.19c	81.84 ± 15.31s	18.65 ± 0.09f	27.92 ± 0.08h	35.53 ± 7.00g	0.13 ± 0.03f	4.19 ± 0.93g
Minimum	263.31	nd	6.24	2.22	nd	nd	Nd
Maximum	3358.65	2045.59	35.43	495.15	93.26	0.72	33.98

Results are given as means ± standard deviation. Means followed by the same letter in the column do not differ statistically from each other by Scott-Knott test (P ⩽ .05).

L-tryptophan.

5-Hydroxy-tryptophan.

Tryptamine.

Serotonin.

Melatonin.

L-DOPA.

Dopamine.

L-Trp levels ranged from 263.31 µg/100 g (2A4, Spring 2018) to 3358.65 µg/100 g (1B2, Spring 2019) (Figure 2A; Table 3). This difference is attributed to the plant species visited by the bees during nectar collection, as well as the honeys’ geographic origin and the time/season of harvest. Other studies highlight L-Trp values greater than 14 mg/kg in rosemary honeys, while levels reached 1.9 mg/100 g in sunflower honeys and 1.10 mg/100 g in lavender honey. Among the samples analyzed, the lowest L-Trp content was found in honey whose main flowering was Hovenia dulcis (Japanese grape) (2A4), from the Atlantic Forest region, with a Köppen climate classification of Cfa and harvested in Spring 2018 and grouped in PC1− and PC2− (Figure 1). It is worth mentioning that, at the same time of harvest, Mel was not detected. These results also demonstrate that the year of harvest and season affects the levels of biogenic amines (Figure 2A and B). On the other hand, the honey with the highest L-Trp content (3358.65 µg/100 g) was produced in the coastal region of Santa Catarina, whose predominant bloom was composed of wild flowers (polyfloral honey) and harvested in spring 2019 (Table 1; Figure 2A and B). Some articles describe that L-Trp content varies depending on the predominant flora. Hermosín et al detected L-Trp values of between 0.19 and 1.10 mg/100 g and the variation was dependent on the predominant flowering, that is, the lowest content was found in orange blossom honeys and the highest in lavender honeys. The authors claim that amino acid composition does not exactly distinguish the botanical origin of honeys, or their authenticity. In our study, the L-Trp content of most samples is very close to that found in the literature and sample harvested in spring showed the highest level of this amino acid; however, it is worth noting that the botanical origin, as well as the climate, region, and season, can affect the level of metabolites, including amino acids. The levels of metabolites formed from tryptophan are not correlated with the results of L-Trp content.

L-Trp found in honeys may come from plants (pollen, nectar, and resins) or from the metabolism of bees during honey production. In pollen, the contents of this aromatic amino acid are quite variable and may be very low, that is, 0.028 g/kg to 0.197 g/g[18,19] or much higher, such as described in Rhododendron ponticum pollen (8053.00 µg/g).

L-Trp in honey has been the subject of several studies related to human health. For example, L-Trp supplementation appears to improve the social behavior of people suffering from disorders due to malfunctioning of the serotonergic system.[21,22] Intake of L-Trp has been linked to decreased levels of psychosis in both animal and human studies and sleep deprivation. Other studies have demonstrated that ingestion of honey with milk before bedtime may decrease hypoglycemic effects in diabetic patients, or improve the sleep quality of healthy people or those with coronary heart problems. Thus, L-Trp supplementation appears to improve control over social behavior in individuals who suffer from disorders or behaviors associated with dysfunctions in serotonergic functioning.

L-Trp is transported into the brain via the leucine-preferring L1 system and may compete with other amino acids (eg, tyrosine, phenylalanine, leucine, isoleucine, and valine) called “large neutral amino acids” (LNAA). The L-Trp : :p : LNAA ratio determines the flux of this amino acid and, consequently, the biosynthesiSer in the brain. Foods with a high content of L-Trp, such as honey, often also contain other amino acids in varying concentrations. Thus, the net effect of the L-Trp content in honeys is relevant, but it should be considered carefully with regard to possible increases in Ser synthesis, given the competition for a transporter system in the blood–brain barrier between this amino acid and the other LNAA. Furthermore, excess L-Trp may cause adverse reactions, such as gastric problems and dizziness, among others.

The remainder of the L-Trp that is not used for protein synthesis may be converted to biomolecules such as kynurenine (KYN) (responsible for approximately 90% of L-Trp catabolism ) or those related to neuroimmunological signaling, such as Ser and Mel. About 1% of dietary L-Trp is used for Ser synthesis,[26,27] by the conversion of L-Trp to 5-OH-Trp or Tryp, depending on the metabolic pathway. KYN, derived from L-Trp catabolism, originates niacin, precursor of the coenzyme nicotinamide adenine dinucleotide (NAD+). In this pathway, 60 mg of Trp produces 1 mg of nicotinic acid or niacin. The usual intake of Trp is approximately 900 to 1000 mg per day, and the recommended daily dose is around 4 mg/kg body weight per day for adults and 12 mg/kg body weight for children. According to our results, the consumption of honey as a source of L-Trp may help in several metabolic processes, including psychiatric and neurological disorders and anticancer immunity. In children and adolescents with autism spectrum disorder, the intake of L-Trp may decrease symptoms of irritability and mild depression due to increased Ser levels.

Although there is no consensus on the necessary amount of 5-OH-Trp or Tryp (Ser precursors) to be ingested daily, considerable levels were detected in some honeys as 5-OH-Trp (4B3-2045.59 µg/100 g) and Tryp (2A6-35.01 µg/100 g and 2C3-35.43 µg/100 g) (Table 3). Foods with higher levels of 5-OH-Trp and Tryp may favor the formation of Ser and Mel, due to the ease in crossing the brain–blood barrier (BBB), since both the synthesis and the turnover of Ser depend on the intake of L-Trp and 5-OH-Trp. In this study, we highlighted the maximum content of 5-OH-Trp (2045.59 µg/100 g) in the sample from Baccharis dracunculifolia DC (4B3), originating from an altitude of approximately 900 m. The same compound was detected at a lower level (22.99 µg/100 g) in 2A4, from a lower altitude (Alto Vale do Itajaí), that is, 593 m, which also showed a lower content of L-Trp (263.31 µg/100 g) and Tryp (6.24 µg/100 g) and no melatonin, L-dopa and dopamine (Table 3). Tryp has been used as a fermentation marker, along with other amines such as tyramine, cadaverine, putrescine, and histamine. This aromatic and heterocyclic biogenic amine can induce vasoactive or psychoactive effects on the human body. According to the authors, the consumption of 25 to 30 mg/kg of Tryp can cause migraines; however, to reach these values, it would be necessary to consume approximately 900 g of honey from the samples that contain the highest levels.

Ser cannot cross the BBB and its intake contributes to a decrease in reactive oxygen species, as described in red blood cells of a mouse model, mainly in the process of lipid peroxidation. In the present study, Ser, Dop, and the amino acid L-dopa were all detected at higher levels in honeys from the mild Cfb climate. In the present study, Ser was detected in greater amounts (495.15 µg/100 g) in 4B6, a honey from an altitude of 1280 m, produced in spring 2018 and Cfb climate (Table 3). In this region, “aroeira” (Schinus sp.), “aroeira branca” (Lithraea molleoides), and “caúna” (Ilex theezans Mart. ex Reissek) are predominant (Table 1). The non-detection of Mel stands out in this sample. The lack of conversion of Ser into Mel could eventually contribute to an increase of Ser in the investigated honey samples. However, this was not observed in samples that did not show detectable levels of Mel. A low Ser content was verified in honeys from 2A2 and 2B2, both from a Cfa climate and from the Atlantic Forest ecosystem, but from different flowerings, regions, and altitudes (Table 3).

Higher levels of Mel, unlike Ser and Dop, were detected in honeys from coastal regions (agroecological regions coast of Florianópolis and Laguna, 1B4-93.26 µg/100 g) with a Cfa climate, low altitude (92 m), predominantly flowering of Eucalyptus and harvest in Spring 2019. Mel, Ser, and Dop were grouped in PC1− (Figure 2A) according to the year of harvest and in PC1+ in relation to the season (Figure 2B). In these figures and in Table 3 it is possible to verify that the levels of Mel varied between seasons, year, flowering, and climate and it is not possible to use this compound as a biochemical marker of honey quality. Mel, unlike Ser, crosses the BBB, in addition to having high antioxidant potential and not being able to be stored in the pineal gland, being released into the bloodstream and rapidly degraded in the liver. Mel is an essential molecule related to the circadian rhythm; it has immunomodulatory and neuroprotective actions in tumor suppression, in addition to being related to oxidative stress. Several studies were carried out during the Covid-19 pandemic using Mel and it has been recommended that the use of this substance may be related to a decrease in side effects due to its anti-inflammatory and antioxidant action.[35,36] Thus, honeys with higher levels of Mel could be an interesting source of this amine, given its already demonstrated pharmacological effects.

In the studied honeys, the presence of L-dopa and Dop was also evidenced, the levels reaching 0.72 and 33.98 µg/100 g (4B6), respectively (Table 3), in honey from Vale do Rio do Peixe and Alto Irani with an altitude 973 m, Cfb climate and flowering of “aroeira” (Schinus sp.), “aroeira branca” (L. molleoides), and caúna (I. theezans Mart. ex Reissek) (Table 1). It is important to mention that the highest Ser content was also detected in these samples.

L-dopa levels in honeys are poorly described in the literature, which makes the data found in this work interesting. L-dopa occurs in plants as it is a precursor of several alkaloids, catecholamines, and melanin. In honeys from Turkey, whose predominant flowering was fava beans (Vicia fava), Topal et al reported an L-dopa content of 0.0321 mg/10 g, much higher than those found in honeys from Santa Catarina, Brazil. The L-dopa content may be due to the botanical source, as its presence at considerable levels in several plant species has already been described, including in fava bean genotypes, in flowers, leaves, and fruits. In humans, L-dopa has been used in the treatment of Parkinson’s disease, characterized by a deficiency in the synthesis of this catecholamine and as Dop cannot cross the BBB, while L-dopa does and is decarboxylated to form Dop in nerve cells. In addition to acting as a neutrotransmitter, Dop may act as an immunomodulatory regulator, besides being indispensable for neuroimmune communication, i.e., its relationship with alterations in the functions of macrophages, lymphocytes, neutrophils and monocytes, showing that immune cells, in homeostatic and pathological conditions, interact with Dop centrally and peripherally. Some studies have shown a relationship between Dop levels and Dop receptors and diseases such as glaucoma, diabetes and cardiovascular disease. The analyzed honeys have been demonstrated to be a source of both substances and may be beneficial as adjuvants in therapies aimed at increasing the content of L-dopa and Dop.

Conclusion

The amino acid and biogenic amine content of floral honeys vary depending on several factors, including the agroecological region of production and collection, as well as the nectar donor plants and season. Ser and Dop, as well as the amino acid L-dopa, showed a significant and strong correlation and were detected in higher levels in honey from agroecological regions with a milder climate (Cfb), at higher altitudes and in Spring 2018. A higher content of 5-OH-Trp was also found in samples from a milder climate, harvest in 2019. On the other hand, L-Trp and Tryp, as well as Mel, were found at higher levels in honeys harvested in 2019 during the hottest seasons and in Cfa climate.