A comparative study of the antihyaluronidase, antiurease, antioxidant, antimicrobial and physicochemical properties of different unifloral degrees of chestnut (Castanea sativa Mill.) honeys

Abstract

This study was planned to investigate some physicochemical and anti-inflammatory, antioxidant, antimicrobial properties of three different degrees of unifloral characters of chestnut honeys. Antihyaluronidase, antiurease and antimicrobial activities were evaluated as anti-inflammatory characteristics. Total phenolic contents, flavonoids, tannins, phenolic profiles, ferric-reducing antioxidant power (FRAP), scavenging activities of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS⁺) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals were evaluated as antioxidant properties. Color, optical rotation, conductivity, moisture, pH and ash content were evaluated as physicochemical parameters, and some sugars content, prolin, diastase, HMF and minerals (Na, K, Ca, P, Fe, Cu and Zn) were evaluated as chemical and biochemical parameters. All studied physicochemical and biological active properties were changed in line with the unifloral character of the chestnut honeys. A higher unifloral character was found associated with greater apitherapeutic capacity of the honey, as well as biological active compounds.

Keywords:

• Antioxidant
• chestnut
• honey
• hyaluronidase
• inhibition
• phenolics
• urease

Introduction

Chestnut (Castanea sativa Mill.) trees are abundant in central and southern Europe, as well as around the Mediterranean basin (e.g. North Africa). Chestnut flowers are one of the best sources of nectar and pollen for honeybees at the beginning of summer1. Trees are also cultivated in the north of Turkey, especially around the Black, Marmara and Aegean seas2. With its color ranging from light amber to dark and woody taste and aroma, chestnut honey differs significantly from blossom honeys1,3. This honey, which does not crystallize easily, is widely used in the treatment of colds, reflux, gastritis and other disorders of the upper respiratory tract and gastric system4. The honey consists largely of carbohydrate (75–76%) and water (16–21%), with very small amounts of various proteins, amino acids, polyphenols, vitamins organic acids and minerals5–7. Secondary metabolites comprising approximately 2% of the honey content, such as essential oils, polyphenols, vitamins, alkaloids, organic acids, amino acids and proteins, are largely responsible for its sensory, aromatic and active biological properties2,5,7. Polyphenols consist of some subclasses, such as phenolic acids, flavonoids, tannins, stilbenes and lignins, the natural compounds are important formation of many sensory properties such as aroma, taste, color, texture and biologically active properties10. The many active biological properties of honey, which vary depending on the type of honey, such as its antioxidant, anti-inflammatory, immunomodulator, antiviral, antimicrobial and antitumor activities, are thought to derive from differences in these components8,9. Previous studies about chestnut honeys have showed that the honey have important antioxidant10, antimicrobial11, antihepatoprotective12, immunomodulator6 and antitumoral13 properties, which is reported to possess high levels of apitherapeutic and biologically active properties. And also, the chestnut honey contained a valuable among of phenolic substances that were responsible for its biological active features14. There are many chestnut honey studies in the literatures, but there are no studies of its unifloral characterization with its associated biologically active properties2,7,10,12,15. For this reason, this study was intended to investigate some physical, chemical and biological active properties of chestnut honeys containing three different levels of nectar sources.

Materials and methods

Reagents

Phenolic standards; gallic acid, protocatechuic, p-OH benzoic, vanillic acid, catechin, syringic acid, ferulic acid, trans-cinnamic acid, rutin, luteon, sugar standards; fructose, glucose, sucrose, maltose, trehalose, melebiose and melezitose) were obtained from Sigma-Aldrich (Munich, Germany). Folin–Ciocalteau reagent was obtained from LiChrosolv® (Merck KGaA, Darmstadt, Germany). 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS^·+), (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) Trolox®, 5-hydroxymethlfurfural (HMF), bovine testes hyaluronidase (400–1000 U/mg solid), Jack bean urease, thiourea, hyaluronic acids sodium salt, bovine serum albumin (BSA), sodium phosphate dibasic (Na₂HPO₄), sodium phosphate monobasic monohydrate (NaH₂PO₄.H₂O), gallic acid, sodium carbonate (Na₂CO₃), sodium acetate trihydrate (NaCH₃COO.3H₂O) and vanillin were purchased from Sigma-Aldrich Co. (St Louis, MO). All solvents were HPLC grade degrees. Acetonitrile, methanol and ethanol were supplied by Sigma-Aldrich Co. (St. Louis, MO) and methanol by Merck KGaA, Darmstandt, Germany).

Samples

Thirty-two different chestnut honeys were collected from experienced beekeepers from three different regions of the Black Sea region of Turkey in 2014. Following botanical identifications, the honeys were divided into three groups. Identification of the samples was based on the predominant pollen percentage as a melissopalynological test. Percentages ranged between 46% and 92%. Honeys were considered unifloral or monofloral when the predominant pollen constituted 45% or more of the total pollen grains16. After pollen analyses, the samples were divided into three groups. Table 1 shows the predominant pollen percentages and the groups of the honey samples.

Table 1. Anti-inflammatory activities and some antioxidant parameters of the chestnut honeys.

	G1	G2	G3	Thiourea	Trolox
Antihyaluronidase (IC50 mg/mL)	76.10 ± 3.80*	84.80 ± 3.81^†	129.30 ± 5.17^‡	—	—
Antiurease (IC50 mg/mL)	12.36 ± 0.61*	16.58 ± 0.83^†	34.20 ± 1.54^‡	12.09 ± 0.08	—
Total phenolics (mg GAE/100 g)	94.05 ± 4.70^†	84.50 ± 3.38*	76.20 ± 4.95*	—	—
Total flavonoids (mg QUE/100 g)	6.50 ± 0.39^‡	5.90 ± 0.26^†	4.20 ± 0.21*	—	—
Total tannin (mg tannin/100 g)	16.23 ± 0.81^‡	12.15 ± 0.61^†	10.20 ± 0.77*	—	—
FRAP (μmolFeSO4ċ 7H2O/100 g)	462 ± 18.48^†	392 ± 23.52*	355 ± 23.08*	—	—
ABTS^·+ SC50 (mg/mL)	14.241 ± 0.64*	17.380 ± 0.87^†	34.761 ± 1.21^‡	—	0.181 ± 0.001
DPPH SC50 (mg/mL)	21.20 ± 1.06*	27.06 ± 1.78^†	38.20 ± 1.91^‡	—	0.004 ± 0.001

The superscripts *, † and ‡ are significantly different at the 5% level in the same columns (p <  0.05). IC₅₀ value of thiourea was given in terms of μg/mL and trolox was given in terms of mg/mL.

Extraction for biological tests

Methanolic honey extracts were prepared in order to determine antioxidant capacity. For extraction, 25 g of crude honey sample was placed in a falcon tube (100 mL), and 50 mL 99% methanol was added. The mixture was continuously stirred with a shaker (HeidolphPromax 2020, Schwabach, Germany) at room temperature for 12 h. Particles were removed with Whatman filter paper. The final volume of the solution was adjusted with methanol to 50 mL. The methanolic extract was then used for all antioxidant purposes.

Antihyaluronidase activity

Antihyaluronidase activity was measured against bovine testes hyaluronidase as a spectrometric assay. The assay was based on the Sigma protocol with slight modifications17. The reaction mixture consisted of 100 μL of hyaluronidase (1.67 U/mg), 100 μL of phosphate buffer (200 mM, pH 7.37 °C) with 77 mM sodium chloride and 0.01% BSA mixed with 25 μL sample extract solution. After preincubation at 37 °C for 10 min, the reaction was initiated by the addition of 100 μL of substrate solution in the form of hyaluronic acid (0.03% in 300 mM sodium phosphate, pH 5.35) and incubated at 37 °C for 45 min. The undigested hyaluronic acid was mixed with 1 mL acid albumin solution made up of 0.1% bovine serum albumin in 24 mM sodium acetate and 79 mM acetic acid, pH 3.75. After the mixture was left at room temperature for 10 min, the absorbance was measured at 600 nm using a Thermo Scientific Evolution 260 spectrophotometer (Thermo Scientific, Waltham, MA). The IC₅₀ value was determined as the concentration of compound producing 50% inhibition of maximal activity.

In order to calculate the half maximal inhibitory concentration (IC₅₀), a series of concentrations and their response data are required (e.g. sample concentrations X1, X2, … , Xn and % inhibition based on absorbance changing Y1, Y2, … , Yn).

Percentage inhibition of the sample is calculated using the equation

The inhibition values of Y are in the range 0–100. For concentration–response data, a logarithmic function may be used instead of linear regression. After adding the response curve as logarithm-transformed, x values as IC₅₀ are calculated using the following equation: (I) (II)

Antiurease activity

The urease-inhibiting effect of honey was monitored in the form ammonia production18. First, 200 μL of Jack Bean urease, 500 μL buffer (100 mM urea, 0.01 M K₂HPO₄, 1 mM EDTA and 0.01 M LiCl, pH 8.2) and 100 μL aquatic honey sample was incubated at room temperature for 20 min. The phenol reagent (550 μL, 1% w/v phenol and 0.005% w/v sodium nitroprusside) and alkali reagent (650 μL, 0.5% w/v sodium hydroxide and 0.1% v/v NaOCl) were added. The increasing absorbance was read at 625 nm (Thermo Scientific, Waltham, MA) after 50 min. The standard used was thiourea and the results were evaluated as IC₅₀ values, the level producing 50% inhibition of maximal activity.

Determination of total phenolics

Three different total phenolic assays, total phenolic contents (TPC), total flavonoids (TF) and condensed tannin (CT) were employed. Total phenolic compounds were measured using Folin–Ciocalteu assay19. First, 680 μL distilled water, 20 μL methanolic honey extract and 400 μL of 0.2 N Folin–Ciocalteu reagent were mixed in a test tube and well vortexed. Next, 400 μL Na₂CO₃ (7.5%) was added and incubated for 2 h at room temperature. After incubation, the absorbance was measured at 740 nm. TPC was expressed as milligram of gallic acid equivalents per 100 g honey samples. All measurements were performed in triplicate.

Determination of total flavonoids

Total flavonoid concentration was measured using a spectrometric assay20. Briefly, 0.5 mL samples, 0.1 mL of 10% Al(NO₃)₃ and 0.1 mL of 1 M NH₄.CH₃COO were added to a test tube and incubated at room temperature for 40 min. Absorbance was then measured against a blank at 415 nm. Quercetin was used for the standard calibration curve. The total flavonoid concentration was expressed as mg of quercetin equivalents per 100 g sample.

Determination of condensed tannins

Condensed tannins in the extracts were measured with a small modification21. An aliquot (25 μL) of honey extract was mixed with 750 μL of 4% vanillin and dissolved in methanol. Next, 375 μL of HCl (m/v, 37%) was added. The solution was incubated at room temperature in the dark for 20 min. The absorbance against a blank was read at 500 nm. (+)-Catechin was used to make the standard curve (0.05–1 mg/mL). The results were expressed as milligram catechin equivalents (CE)/100 g sample.

Determination ferric reducing/antioxidant power (FRAP)

Ferric tripyridyltriazine (Fe-III-TPTZ) complex was used to determine total antioxidant capacity22. Briefly, working FRAP reagent was prepared as required by mixing 25 mL of 300 mM acetate buffer, pH 3.6, with 2.5 mL of 10 mM TPTZ solution in 40 mM HCl and 2.5 mL of 20 mM FeCl₃·6H₂O solution. Next, 3 mL freshly prepared FRAP reagent and 100 μL of extract were mixed and incubated for 4 min at 37 °C, and the absorbance was read at 595 nm against a reagent blank containing distilled water. Trolox was used as positive control to construct a reference curve (62.5–1000 μM). FRAP values were expressed as μM Trolox equivalent of 100 g sample.

Free radical-scavenging activity of DPPH

About 750 μL of methanol extract was mixed with 750 mL of 0.1 mM DPPH dissolved in methanol, well vortexed and then incubated for 50 min in the dark; absorbance was read at 517 nm. The results were expressed as SC₅₀ (mg/mL), which was calculated from the curves by plotting absorbance values, with SC₅₀ values representing the concentration of extract (mg/mL) required to inhibit 50% of the radicals23.

Free radical-scavenging activity of ABTS

Monocation radical of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^*+) is generated by the oxidation of ABTS with potassium persulfate and is reduced in the presence of such hydrogen-donating antioxidants24. ABTS was dissolved in deionized water to 7 mM, and potassium persulfate was added to 2.45 mM. The reaction mixture was incubated at room temperature overnight (12 h) in the dark. The mixture was then diluted with 0.01 M PBS (phosphate-buffered saline), pH 7.4, to give an absorbance value of ∼0.70 at 734 nm. Trolox was used as standard, and the values were expressed as SC₅₀ (mg sample per mL), the concentration of samples causing 50% scavenging of ABTS radicals.

Determination of phenolic profile

Thirteen standards of phenolic compounds were analyzed using HPLC (Elite LaChrom Hitachi, Japan). The sample was injected into the HPLC system with a reverse phase C18 column (150 mm × 4.6 mm, 5 μm; Fortis). Acetonitrile, water and acetic acid were used for the mobile phase by applying the programed gradient. The mobile phase consisted of (A) 2% acetic acid in water and (B) acetonitrile: water (70:30). The samples injection volume was 25 μL, column temperature at 30 °C and flow rate at 1.5 mL/min. The programed solvent used began with a linear gradient held at 95% A for 3 min, decreasing to 80% A at 10 min, 60% A at 20 min, 20% A at 30 min and finally 95% A at 50 min5,25.

Determination of antimicrobial activity

All test micro-organisms were obtained from ATCC and RSKK were reconstituted from its lyophilized form according to the manufacturer’s protocol, were as follows: E. coli ATCC (25922), Y. pseudotuberculosis ATCC (911), P. aeruginosa ATCC (43288), S. aureus ATCC (25923), E. faecalis ATCC (29212), B. cereus(709 Roma), M. smegmatis(ATCC607), C. albicans ATCC (60193), S. cerevisiae (RSKK 251). Microbial bacterial cultures were grown in Mueller Hinton Agar (Merck) and broth, M. smegmatis ATCC607 was grown in Mueller Hinton agar and broth (Merck) and S. cerevisiae and C. albicans were grown in potato dextrose agar (Difco) and yeast extract broth. Antimicrobial activity of the extracts was determined using the disc diffusion method26. Bacterial suspension turbidity 0.5 McFarland and fungal suspension turbidity 2.0 McFarland standards were prepared. The concentration of bacterial suspensions was thus adjusted to 10⁷cells/ml, and that of fungal suspension to 10⁸ cells. Sterile swabs were immersed in the test organism. Excess fluid was removed, and the inoculated was applied to the entire surface of the plate in at least three directions. Discs were applied to the plate within 15 min of inoculation. Solutions 1/5 μL in size were impregnated on 6-mm-diameter sterile blank discs (Oxoid) (20 μL per disc). Inhibition zones were measured using digital calipers after incubation for 24 and 48 h at 37 °C (for bacteria). The results were expressed in terms of the diameter of the zones;  <6 mm, inactive; 7–9 mm, very low activity; 9–11 mm, low activity; 12–14 mm, average activity and  >15 mm, high activity. Ampicillin, streptomycin and fluconazole were used positive controls, and all determinations were made in triplicate.

Physicochemical properties of chestnut honey

Analyses of all physical characteristics of the honeys were performed in line with European Union legislation27. Moisture was measured using a refractometer (Atago Refractometry, Tokyo, Japan), electrical conductivities using a conductometer (WTW inoLab Cond/720conductimetry, Germany), and optical rotation using a polarimeter (Beta PPP7, England). Color parameters of the honeys were measured using a Hunter spectrometer (CR-400, Minolta, Osaka, Japan).

Hydroxymethylfurfural (HMF) was determined with HPLC-UV (Elite Lachrom Hitachi, Japan) using a C₁₈LiChroCART® column 250– 4 RP (10 μm)28. Prolin content was measured using spectrometric assay24. Diastase activity was assayed with spectrometric assay using the soluble starch method at 40 °C22. The diastase units were expressed as 1% starch hydrolyzed by an enzyme in 1 g of honey in 1 h.

Determination of sugar profile

Seven different sugar standards were analyzed using high-performance liquid chromatography (HPLC) a with refractive detector (RID)(200/4.6 Nucleosil 100–5 NH₂, Elite LaChrom, Hitachi, Japan). Standards of sugars were dissolved (1 g/10 mL) in deionized water and filtered with HPLC syringe filters (0.45 μm). The calibration curves were between 0.994 and 1.000. Seventy-nine percent acetonitrile and 21% water were used as mobile phase in the isocratic elution program5. Injection of sample volume was 25 μL, column temperature was 40 °C and flow rate was 1.5 mL/min.

Determination of mineral profile

ICP-MS (Agilent, 7500cx) was used to measure minerals in the honey samples. Honeys (approximately 1 g) were digested with a Teflonmicro-digestion vessel (CEM, MARSXperss) to which were added 5 mL high purity nitric acid and 1.5 mL 30% H₂O₂. The samples were digested at 120 °C for 45 + 25 min, held at 190 °C 30 + 10 min, allowed to cool and then made up to 14 mL with distilled water. For calibration standards for measurement with ICP-MS, Merck VI multielement calibration solution was prepared volumetrically by dilution with 1% nitric acid. The lowest detection concentration was 0.0002 mg/L29.

Statistical analyses

All statistical analyses were performed on SPSS 16.0 for Windows (SPSS Inc., Chicago, IL) software. Results are given as mean values and standard deviation. Differences between the honey groups were analyzed using the independent samples t-test.

Results and discussion

Thirty-two different chestnut honeys were collected from different areas in the Black Sea region of Turkey. Although all the honey samples were unifloral in nature, we classified them into three different groups, high unifloral (Group 1), medium unifloral (Group 2) and low unifloral (Group 3). Table 1 shows the pollen analyses and dominant pollen percentages of the samples. The pollen percent of the samples were changed between 46% and 95% (Table 5). Honeys were considered unifloral or monofloral when the predominant pollen constituted 45% or more of the total pollen grains16.

The anti-inflammatory capacities of honeys were measured by determining hyaluronidase and urease enzyme inhibitions. Hyaluronic acid (HA), an important glycosaminoglycan in the extracellular matrix and connective tissue, is degraded by the enzyme hyaluronidase30. Active hyaluronidase caused HA deficiency, and the breakdown products of this polysaccharide are also harmful to the organism. Inhibitors of the enzyme have a high potential for therapeutic use in several diseases, such as cancer, rheumatoid and arthritis31,32. Novel natural inhibitors are needed in many areas to contribute to the controlled inhibition of hyaluronidase. However, hyaluronidase inhibitors have also led to new therapeutic concepts in pathophysiological conditions that associated with the hyaluronan–hyaluronidase system9.

Chestnut honeys inhibited the hyaluronidase at varying IC₅₀ values (76 to 129 mg/mL), (Table 1) and this inhibition was associated with their unifloral character. In our previous study, it was found that HAase inhibition was related to the honeys’ phenolic contents; the higher the phenolic content in the honey’s, the greater the inhibitory effects.

Urease is an enzyme that catalyzes the hydrolysis of urea into carbon dioxide and ammonia33. Ureases that produce excess ammonium are also involved in the development of urolithiasis, pyelonephritis, hepatic encephalopathy, hepatic coma urolithiasis and urinary catheter encrustation in humans and animals9. The urease inhibition is also important to deal with Helicobacter pylori in gastric diseases9. Helicobacter pylori, an anaerobic bacterium, caused numerous inflammations that can cause gastritis, gastric ulcer, peptic ulcer, reflux and eventually cancer by settling in the gastric mucosa, stomach and duodenum. For survival, the bacteria in the gastric systems need urease enzyme34. Since the bacterium is prevented from adhering in the gastric system with urease inhibition, urease inhibitors are particularly important in the treatment of gastric ulcers35. The chestnut honey has been shown to inhibit the urease with ranging from 12 to 34 mg/mL inhibition concentrations (IC₅₀) (Table 1) and that inhibition values was related with unifloral character of the honey. It was reported that chestnut honey inhibition urease with 10–34 mg/mL inhibition values35, that the results were similar to our results. In traditional uses in Turkey, chestnut honey is to be used especially in the treatment of gastric disorders, may be the treatment progresses is based on the urease inhibition. All groups of the chestnut honeys in the study inhibited hyaluronidase and urease in a manner dependent on the varying IC₅₀ values and concentrations. G1 honeys exhibited the highest degree of inhibition of hyaluronidase and urease, followed by G2 and G3. In conclusion, both enzymes were inhibited by the chestnut honeys depending on the honeys’ unifloral character, and honeys with high pollen and associated high polyphenol contents possessed greater biological activity. G1 samples were distinguished from other G2 and G3 code honeys, having the highest total phenolics, total flavonoids, condensed tannin, most phenolic compounds and it showed the highest inhibition effect (the lowest IC₅₀ value) against hyaluronidase and urease enzymes.

The great majority of the biologically active characteristics of honey derive from the secondary metabolites in its matrix. The relation between the bioactive compounds in chestnut honey, which is reported to be rich in phenolic contents, and the unifloral property of the honey has been investigated in previous studies5. The phenolic compounds in honey were measured as total phenolic content (TPC), total flavonoid (TF) and total condensed tannin (TCT) levels (Table 2). Levels of phenolic materials ranged between 70 and 105 mg GAE/100 g, those of TCT between 9.12 and 17.02 mg catechin/100 g and those of TF between 4.04 and 7.01 mg QUE/100 g. Concentrations of compounds from all three polyphenol classes varied depending on the unifloral character of the chestnut honey (Table 1). The great majority of the phenolic compounds of the chestnut honey in the study derived from condensed tannins. Tannins are phenolic polymers consisting of multiple anthocyanin-like molecules37. Phenolic compounds were also analyzed by exposure to acid hydrolysis in order to reveal total tannin or condensed flavonoids in the chestnut honey. Thirteen phenolic components were identified at HPLC in the phenolic profile of chestnut honeys. With the exception of luteon and syringic acid, components were identified at various levels in chestnut honey from all three groups (Table 2). p-OH benzoic, cumaric acid, protocatechuic acid and caffeic acid were identified as the major phenolic compounds among the tested substances. Similarly to our own findings, one previous study reported that chestnut honeys with a major pollen content exceeding 70% were rich in caffeic acid, catechin and chlorogenic acid38. Phenolic acid standard chromatographic (Figure 1) and RP-HPLC-UV validation parameters of phenolic compounds (Table 6). An increase approaching 3-fold was observed in catechin, epicathecin, caffeic acid, vanillic acid and rutin levels with acid hydrolysis in honeys from the second group, while there was no change in the other seven phenolic compounds. The increase in flavonoid levels, in contrast to phenolic acids with the exception of vanillin, at acidic hydrolysis may be attributed to tannins. The characteristic woody aroma and dark color of chestnut honey may derive from the tannin and lignins in its structure. The antioxidant capacities of chestnut honeys were measured using the ferric reducing/antioxidant power (FRAP), DPPH and ABTS radical scavenging activity tests. All three are widely used methods for revealing the antioxidant characteristics of natural and bee products. The values determined are given in Table 1. FRAP values ranged between 330 and 470 μmol FeSO₄/100 g, the antioxidant capacity of honey rising in line with its unifloral character. Honeys’ free radical-scavenging capacities and antioxidant effects were tested with the radicals DPPH and ABTS, the inhibition values of both radicals (SC₅₀ mg/mL) exhibited reverse correlation with the unifloral character of honey. A significant correlation was determined with the radical-scavenging effect determined and honey’s total phenolic contents and FRAP values (Table 3).

Table 2. Phenolic profiles of the chestnut honeys.

Compounds	G1	G2*	G3
Gallic acid	1.58 ± 0.20‡	1.05 ± 0.03*	0.82 ± 0.10†
Protocatechuic acid	6.45 ± 1.25‡	5.05 ± 2.08†	4.40 ± 0.58*
p-OH benzoic acid	12.10 ± 1.10†	8.45 ± 0.80*	2.45 ± 0.61*
Catechin	1.25 ± 0.08‡	0.82 ± 0.04†	0.23 ± 0.01*
Vanillic acid	n.d.	n.d.	n.d.
Caffeic acid	6.40 ± 0.63†	3.60 ± 0.25*	3.01 ± 0.15*
Syringic acid	0.67 ± 0.12	0.50 ± 0.20	0.45 ± 0.04†
Epicatechin	n.d.	n.d.	n.d.
p-Coumaric acid	5.12 ± 1.13‡	4.40 ± 0.50†	1.55 ± 0.25*
Ferulic acid	2.32 ± 0.50‡	1.78 ± 0.33†	0.40 ± 0.20*
Rutin	2.40 ± 0.12†	0.13 ± 0.01*	0.08 ± 0.01*
t-Cinnamic acid	1.18 ± 0.60*	1.05 ± 0.14*	0.90 ± 0.054†
Luteolin	n.d.	n.d.	n.d.

The superscripts *, † and ‡ are significantly different at the 5% level in the same columns (p < 0.05).

*n.d.: not.

Table 3. Pearson correlations of the biological active compounds and tests of the chestnut honey.

		TPC	TF	TCT	FRAP	ABTS	DPPH		ANTI- HYA	ANTI urease
TPC	Pearson correlation	1	0.932**	0.964**	0.985**	−0.779*	−0.783*		−0.748*	−0.791*
	Sig. (two tailed)		0.000	0.000	0.000	0.013	0.013		0.021	0.011
TF	Pearson correlation	0.932**	1	0.896**	0.897**	−0.943**	−0.910**		−0.921**	−0.945**
	Sig. (two tailed)	0.000		0.001	0.001	0.000	0.001		0.000	0.000
TCT	Pearson correlation	0.964**	0.896**	1	0.987**	−0.791*	−0.848**		−0.775*	−0.810**
	Sig. (two tailed)	0.000	0.001		0.000	0.011	0.004		0.014	0.008
FRAP	Pearson correlation	0.985**	0.897**	0.987**	1	−0.754*	−0.788*		−0.729*	−0.771*
	Sig. (two tailed)	0.000	0.001	0.000		0.019	0.012		0.026	0.015
ABTS	Pearson correlation	−0.779*	−0.943**	−0.791*	−0.754*	1	0.975**		0.998**	0.999**
	Sig. (two tailed)	0.013	0.000	0.011	0.019		0.000		0.000	0.000
DPPH	Pearson correlation	−0.783*	−0.910**	−0.848**	−0.788*	0.975**	1		0.981**	0.982**
	Sig. (two tailed)	0.013	0.001	0.004	0.012	0.000			0.000	0.000
ANTI-HYA	Pearson correlation	−0.748*	−0.921**	−0.775*	−0.729*	0.998**	0.981**		1	0.998**
	Sig. (two tailed)	0.021	0.000	0.014	0.026	0.000	0.000			0.000
ANTI-UREASE	Pearson correlation	−0.791*	−0.945**	−0.810**	−0.771*	0.999**	0.982**		0.998**	1
	Sig. (two tailed)	0.011	0.000	0.008	0.015	0.000	0.000		0.000

*Correlation is significant at the 0.05 level (two tailed).

**Correlation is significant at the 0.01 level (two tailed).

The antimicrobial activities of the chestnut honeys against seven bacteria and two fungi strains were examined, and the results were calculated by measuring the resulting inhibition zones (Table 4). With the exception of fungi, diluted chestnut honey samples exhibited good inhibition effects. The highest inhibition was achieved against pathological bacteria of S. aureus, E. faecalis, Y. pseudotuberculosis and E. coli. In contrast to the other biological activities tests, the antimicrobial activities of the chestnut honeys in the three groups were not particularly affected by their unifloral characters. In general terms, since honey is a good antimicrobial mixture it is used in the treatment of various wounds and burns39. The antimicrobial features of honey are caused by factors such as acidity, osmolarity, H₂O₂, and secondary compounds12.

Table 4. Antimicrobial activity of the chestnut honeys (1/2 diluted) against the microorganisms.

	Microorganisms and inhibition zone (mm)
No	Ec	Yp	Pa	Sa	Ef	Bc	Ms	Ca	Sc
G1	15 ± 1.20‡	14 ± 1.00‡	6 ± 0.50*	15 ± 1.10†	14 ± 0.55‡	12 ± 0.62†	6 ± 0.10†	–	10 ± 0.20†
G2	15 ± 1.00‡	12 ± 0.60†	6 ± 0.32*	16 ± 1.12†	12 ± 0.80†	10 ± 0.45*	–	–	–
G3	12 ± 0.80†	12 ± 0.50†	10 ± 0.40†	12 ± 0.80*	10 ± 0.80*	9 ± 0.60*	6 ± 0.50†	–	–
Amp.	10*	10*	18‡	35‡	10*	15‡	–	–	–
Strep.							35‡	–	–
Flu								25	>25*

The superscripts *, † and ‡ are significantly different at the 5% level in the same columns (p < 0.05).

(–): not detected.

Ec: E. coli; Yp: Y. pseudotuberculosis; Pa: P. aeruginosa; Sa: S. aureus; Ef: E. faecalis; Bc: B. cereus; Ms: M. smegmatis; Ca: C. albicans and Sc: S. cerevisiae.

Table 5. Classification of honey and analyzes of some physical and chemical properties of the chestnut honeys.

	Unit	G1 (High) (n = 12)	G2 (Medium) (n = 10)	G3 (Low) (n = 10)
Pollen% Major familia/Other pollen's familiar		91.05 ± 5.58 Fagaceae/Chestnut sative/Tiliaceae, Rosaceae, Ericaceae, Lauraceae, Lamiaceae, Fabaceae	72.66 ± 6.25† Fagaceae/Chestnut sative/Lauraceae, Tiliaceae, Rosaceae, Ericaceae, Poaceae, Fabaceae, Ebenaceae, Asteraceae,	48 ± 4.45* Fagaceae/Chestnut sative/Ericaceae, Tiliaceae, Ebenaceae, Poaceae, Rosaceae, Asteraceae, Fabaceae, Lamiaceae, Lauraceae, Liliaceae
Water	g/100 mL	17.4 ± 1.04*	19.05 ± 0.95*	18.50 ± 1.38*
Conductivity	mS/cm	1.52 ± 0.06*	1.34 ± 0.07†	1.30 ± 0.06†
pH		5.34 ± 0.27*	4.80 ± 0.19*	5.20 ± 0.34*
Total ash	g/100 g	0.88 ± 0.035†	0.85 ± 0.038†	0.74 ± 0.026*
Optic rotation		−1.79 ± 0.089‡	−2.05 ± 0.123†	−2.40 ± 0.144*
L*		7.52 ± 0.45*	15.2 ± 0.76†	24.36 ± 1.21‡
a*		20.04 ± 1.00*	24.18 ± 1.57†	32.46 ± 1.29‡
b*		12.20 ± 0.79*	15.50 ± 0.77†	28.46 ± 1.28‡
Prolin	mg/Kg	1095 ± 54.75‡	972 ± 72.90†	792 ± 39.6*
HMF	mg/kg	2.10 ± 0.15†	1.05 ± 0.068*	5.40 ± 0.32‡
Diastase	DU	17.45 ± 1.13*	18.56 ± 0.65*	19.20 ± 0.96*
Fructose	g/100 g	41.82 ± 1.46†	38.31 ± 1.91*,†	36.40 ± 2.36*
Glucose	“	22.05 ± 1.10*	20.50 ± 1.23*	22.78 ± 1.14*
Sucrose	“	nd	nd	nd
Maltose	“	0.56 ± 0.028*	0.61 ± 0.03*	1.40 ± 0.09†
Trehalose	“	0.45 ± 0.02†	0.42 ± 0.02^*,†	0.39 ± 0.013*
Melibiose	“	0.8 ± 0.04†	1.4 ± 0.07‡	0.42 ± 0.02*
Melezitose	“	1.44 ± 0.07‡	0.87 ± 0.05†	nd*
F/G	-	1.90 ± 0.11†	1.87 ± 0.09†	1.60 ± 0.08*
Na	mg/kg	52.00 ± 2.60‡	35.20 ± 2.30†	28.30 ± 1.80*
K	“	5125 ± 333‡	4252 ± 212†	2524 ± 126*
Ca	“	463.10 ± 23.15‡	410.08 ± 30.70†	320.24 ± 24.00*
Mg	“	67.10 ± 5.02‡	41.21 ± 2.66†	32.05 ± 2.08*
P	“	71.02 ± 4.61†	70.15 ± 2.45†	56.20 ± 1.96*
Fe	“	4.80 ± 0.16‡	3.90 ± 0.19†	2.50 ± 0.13*
Cu	“	2.30 ± 0.11‡	2.05 ± 0.12†	1.45 ± 0.09*
Zn	“	1.45 ± 0.08*	1.38 ± 0.07*	1.41 ± 0.07*
Mn	“	5.84 ± 0.29†	5.60 ± 0.20†	4.36 ± 0.15*

The superscripts *, † and ‡ are significantly different at the 5% level in the same columns (p <  0.05).

The physicochemical characteristics, chemical analyses and mineral contents of the honeys are shown in Table 5. Honeys’ mean moisture content was 18.32%, ranging from 17% to 19%. No correlation was determined between moisture content and pollen levels. Honeys’ electrical conductivity values ranged between 1.20 and 1.55 mS/cm, with a mean value of 1.39 mS/cm. Conductivity in honeys increased in proportion to chestnut pollen levels (r²: p < 0.01). The conductivity of honey derives from the various ions, organic acids, proteins and amino acids and mineral substances it contains. In fact, the conductivity reported for light-colored honey in the honey indices ranges between 0.2 and 0.8 mS/cm, while some honeydew honeys and dark honeys exhibit conductivity values above those limits5. Elevation in chestnut honey conductivity is associated with its high mineral content and related high ash levels. Indeed, the ash content of chestnut honey exceeding that of light-colored floral honey corroborates a mean conductivity of 0.82%10. Values for optical rotation, the ability to rotate polarized light, of chestnut honeys were negative, ranging between −1.79 and −2.40. Optical rotation values being negative (levorotatory) in blossom honeys and positive (dextrorotatory) in honeydew honeys means that rotation constitutes a differentiating physical parameter27,40. The pH values of honeys ranged between 4.80 and 5.34 and no significant difference was determined between them.

Figure 1. Phenolic acid standard chromatographic. 1. Gallic acid, 2. Protocatequic acid, 3. p-OH benzoic acid, 4. Catechin, 5. Vanilic acid, 6. Caffeic acid, 7. Syringic acid, 8. Epicatechin, 9. p-Cumaric acid, 10. Ferulic acid, 11. Rutin, 12. t-cinnamic acid, 13. Luteolin.

Honey color was measured based on the Hunter L a*b* method. In tritium color parameters, the L (0 black-100 white) value expresses the darkness or lightness of honey; a* values express – greenness and  + redness, while b* values express – blueness,  + yellowness. All three color parameters (L, a*b*) in chestnut honey varied depending on the quantities of pollen they contained (Table 5). As the percentage of pollen in chestnut honey increased, as the unifloral property increased in other words, the honey became darker in color (increased L value), and there was an increase in redness (increasing a value) and yellowness (increasing b value). L values reported for blossom in the literature generally range between 50 and 1005,41.

Carbohydrate comprises nearly 95% of dry weight of honey, and carbohydrates consist of 95% fructose and glucose16. The dominant sugar in chestnut honey is fructose, the levels of which in the three groups ranged between 36.40% and 42.82%. Glucose levels ranged between 20.50% and 22.78%. No sucrose was found in any honey, while maltose, trehalose, melezitose and melebiose were detected at levels lower than 1.50%. The F/G ratio ranged between 1.60 and 1.90, decreasing as the unifloral property of honey increased (Table 5). The high fructose content of chestnut honey has been shown to be largely responsible for honey crystallization42.

Table 6. RP-HPLC-UV validation parameters of phenolic compounds.

No	Compounds	R^2	%RSD (Retention Time)	%RSD (Area)	LOD*	LOQ*
1	Gallic acid	0.999	0.210	1.941	0.022	0.067
2	Protocatechuic acid	0.999	0.871	1.920	0.042	0.128
3	p-OH Benzoic acid	0.998	0.351	3.055	0.036	0.109
4	Catechin	0.997	0.492	4.279	0.040	0.121
5	Vanillic acid	1.000	0.828	2.066	0.025	0.075
6	Caffeic acid	0.998	0.179	4.039	0.062	0.187
7	Syringic acid	1.000	0.550	0.848	0.009	0.027
8	Epicatechin	0.999	0.429	3.819	0.030	0.090
9	p-Coumaric acid	0.999	0.204	1.562	0.010	0.030
10	Ferulic acid	0.999	0.222	1.301	0.011	0.033
11	Rutin	1.000	0.234	3.139	0.041	0.123
12	t-Cinnamic acid	1.000	0.262	1.071	0.014	0.042
13	Luteolin	0.994	0.229	5.833	0.043	0.130

*Values are expressed as mg/L.

Mineral analysis with ICP-MS revealed that potassium was the major element in all three group honeys (Table 5). Potassium values represented approximately 50% of the total ash content of honeys. Chestnut honey has been reported to be rich in potassium in previous studies38,43. After potassium, the most abundant elements were calcium and magnesium, the concentrations varying depending on the unifloral character of the honey (Table 5). Mineral levels measured in Slovenian chestnut honey were similar to our study G2 and G3 values, and were reported to be rich in calcium and potassium44.

In conclusion, since chestnut honey is rich in phenolic components and minerals, the honey composition is shaped by its unifloral character, and the higher a honey’s level of chestnut nectar, the higher its biologically active value. Determination of this character, together with the pollen percentages contained in chestnut honey, described as a unifloral honey in the literature, will contribute to a more accurate evaluation of the honey. Since pure chestnut honey is a protective and complementary type it has a high apitherapeutic potential. Regular honey consumption may contribute in reducing inflammatory injury and strengthening human defense systems.

Declaration of interest

We are grateful to the Scientific and Technological Research Council of Turkey (TUBITAK) for its support for the study through its contributions to project No. 114Z370.