INTRODUCTION

Honey is the most consumed apicultural product for nutraceutical and apitherapy purposes worldwide. The nutritional content, antioxidant capacity and sensory such as the colour of honey vary by geographical origin, flora and botanical origin. Especially, the colour and geographical origin generally influence the consumers' preferences and also the sale price of honey (FERREIRA et al., 2009; BODOR; BENEDEK, 2021; PU, 2022). The colour of honey is usually determined by some visual method comparing a standard scale. “Pfund scale” is the most commonly used technique that classifies the colour of honey from water white to dark amber (0-140 mm Pfund) (BODOR; BENEDEK, 2021).

In addition to sensory characteristics, the antioxidant capacity of honey is directly related to phenolic, polyphenol, and flavonoid substances and vitamins such as vitamins C and E in honey which is synthesized by plants. The amounts of these antioxidant compounds vary by the botanical origin of honey which determines its nutritional and sensory characteristics of honey (FERREIRA et al., 2009; MARTINELLO; MUTINELLI, 2021; PUŚCION-JAKUBIK et al., 2022). Vitamin C equivalent is commonly used to determine the total antioxidant capacity level of foods and natural product extracts. The method of Vitamin C Equivalent Antioxidant Capacity (VCEAC) is based on the colorimetric [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay reading by a spectrophotometer. The MTT reagent reacts with the reducing reagents and the antioxidants in the foods or products. And, a dark purple formazan salt is formed as the enzymatic reduction product of MTT (MURAINA et al., 2009; LIU; NAIR, 2010).

The present study was aim to investigate the colour characteristics and antioxidant capacity of honey originating from various botanical and geographical origins in Turkey by using Pfund scale and MTT assays and the interaction between these parameters.

MATERIAL AND METHOD

Honey samples

In this study 8 honey samples were analysed from different geographical and botanical sources: two blossom, two honeydews, one citrus, one thyme-Astragalus, one lavender and one chestnut. All samples were collected from retail markets and originated from different geographical areas of Turkey (Figure 1). Detailed information and the abbreviation and codes of honey sample types were given. The samples were stored in dark room conditions until analysis.

Figure 1
The geographical origins of honey samples 8 samples in Turkey

Determination of honey colour by Pfund scale

For the determination of colour using the Pfund scale, five grams of each sample was dissolved into 10 ml of ultra-distilled water and vortexed well. The samples were heated up to 50 °C in the water bath to dissolve the sugar crystals. After incubation, 100 µl of each sample was added to each well of the microplate in six replicates. The microplate was read at 600 nm to measure the absorbance (nm). The results were evaluated concerning the colour scale (BODOR; BENEDEK, 2021). The Pfund score of all honey was calculated with the equation 1:

mm Pfund = −38.70 + 371.39 × Absorbance (nm) (Eq. 1)

Vitamin C Equivalent Antioxidant Capacity (VCEAC)Vitamin C Equivalent Antioxidant Capacity (VCEAC)

The assay was adapted from the studies of Muraina et al. (2009) and Liu et al. (2010). Briefly, vitamin C (l-ascorbic acid, HPLC grade) was two-fold diluted as 600, 300, 150, 75 and 37.5 µmol with ultra-distilled water. The honey samples were diluted with ultra-distilled water (1:9 w/v). 380 µl MTT (1 mg/ml) and 20 µl of each vitamin C dilation and the samples were mixed in an Eppendorf tube and incubated at 37 °C for 4 h. After incubation, 400 µl DMSO was added to all tubes and mixed well to solve the blue formazan salt formed during the incubation. 100 µl of each mixture was added to the U-bottom 96-well microplate in six replicates. The microplate was read at 570 nm (Absorbance 96, Byonoy, Germany). The standard curve was generated with optic density (OD, nm) values of six vitamin C dilutions by linear regression analysis (vitamin C equivalence = m x OD + b, R²). The VCEAC of the samples were calculated concerning the standard curve.

Data Analysis

The Vitamin C Equivalent of the samples was calculated by the vitamin C standard curve generated with the linear regression between the dilutions of l-ascorbic acid and their optic density values (nm) in Office Excel 2016 (Microsoft, USA). Statistical analysis (one-way ANOVA, Spearman correlation and linear regression) was conducted with SPSS version 15 software (IBM, USA).

RESULTS AND DISCUSSION

The colours of honey samples on Pfınd scale

The absorbance (nm) of the samples was measured in the range from 0.135 to 0.474 nm. Their colour scores on Pfund scale were calculated as from 11.43 to 137.33. The results indicated the colours of eight samples: extra white for two samples (2-Blo-EWh and 7-Citr-EWh), white for two samples (1-Blo-Wh and 6-Lav-Wh), amber for three samples (3-ThyAst-Amb, 4 and 5-Hdews-Amb) and dark amber for one sample (8-Che-DAmb) (Table 1). The highest Pfund score was 137.33 as dark amber in the chestnut honey sample (8-Ches-DAmb) originating from the northern cities of Turkey. The lowest Pfund scores were 11.43 and 13.39 as extra white in the multifloral blossom honey sample (2-Blo-EWh) and the citrus honey (7-Citr-EWh) originating from the Middle, Eastern and Mediterranean coast of Anatolia respectively (Table 1). The honey colour can be differed by beekeepers’ handling method, mineral content, exposure to high temperature and light, and botanical origin of honey (RATIU et al., 2020; ALBU et al., 2021). Many studies reported various Pfund scores for honey originating from different countries. While honey such as multifloral blossom, citrus and acacia generally had the colours of water white, white or extra white, chestnut honey had a darker colour worldwide, also in Europe and Turkey. (KIVRAK et al., 2017; RATIU et al., 2020; ALYGIZOU et al., 2021; SÁNCHEZ-MARTÍN et al., 2022).

Vitamin C Equivalent Antioxidant Capacity (VCEAC)

The vitamin C equivalence of the samples was calculated with regards to the standard curve by linear regression analysis (vitamin C equivalence, µmol =0.00006 x Abs. + 0.0506, R² = 0.9958) (Table 2, Figure 2). Vitamin C equivalents were determined between 412.22 and 1117.78 µmol/mg or 725.99 and 1968.59 µg/mg (Table 2). There was a significant difference between the honey samples (P<0.01). The highest VCEAC was statistically measured in the honeydews honey (5-Hdews-Amb) and the citrus honey (7-Ctr-EWh), the lowest VCEAC was statistically determined in the chestnut honey (8-Che-DAmb) and both blossom honey (1-Blo-Wh and 2-Blo-EWh) (Table 2). There were no significant differences between the honey samples botanically originating from the honeydews (4-Hdews-Amb), the thyme-astragalus (3-ThyAst-Amb) and the lavender (6-Lav-Wh) (P>0.05). Also, there was no significant correlation (β coefficient=0.117; P=0.0585) and regression (R2=0.003, P=0.791) between Pfund scores and VCEAC of the honey samples.

Figure 2
The vitamin C standard curve, Vitamin C Equivalent Antioxidant Capacity (VCEAC) and Pfund scores of honey samples of Turkey (8 samples).

The phenolic, polyphenol, flavonoid and vitamin C contents of honey mostly indicate its antioxidant capacities such as Gallic acid equivalent, ascorbic acid equivalent (AAE) and free radical-scavenging (FERREIRA et al., 2009; MARTINELLO; MUTINELLI, 2021). Many previous studies suggested that the botanical origin, nutritional and colour characteristics directly influenced the amounts of these antioxidant compounds. These studies indicated that the honey had 21.9-58.21, 17.8-165.7, 9.5-17.8 and 0.8-28.2 mg AAE/100 g honey harvested from Turkey (Rhododendron), Italy (honeydews), Spain (chestnut, honeydews, multifloral) Brazil (Meliponinae) respectively (SILICI et al., 2010; ESCUREDO et al., 2013; ATTANZIO et al., 2016; BILUCA et al., 2016, 2020). In this study, ascorbic acid equivalents were measured between 7.26 and 19.69 mg/100g honey expressed as VCEAC by MTT assay in harmony with the results of previous studies.

Ferreira et al. (2009) suggested that the phenolic, flavonoids, ascorbic acid (14.5 mg/ 100g honey), beta-carotene contents, DPPH scavenging and TBARS activity increased by getting darker the colour of honey. Thus, the colourization and pigmentation in honey positively correlated with physicochemical parameters, nutritional content, phenolic compounds and flavonoids of honey (TSIGOURI et al., 2004; EL SOHAIMY et al., 2015; MALDONADO et al., 2021). Moreover, consumer preference and the price of honey are influenced by sensory characteristics (Bodor; BENEDEK, 2021; PU, 2022). But, no significant correlation and regression were determined between Pfund scores and VCEAC of the honey samples in this study.

CONCLUSION

Vitamin C equivalent could be considered as a marker of the antioxidant capacity of honey from different botanical and geographical origins. The highest antioxidant capacity was found in honeydews and citrus honey which has lighter colour than dark amber coloured chestnut honey. So, the honeys originating from Turkey also showed the vitamin C equivalent antioxidant capacity independently of their colour. It might be related and influenced the floral diversity and the phytochemical contents of honey such as phenolic, polyphenol, flavonoid and ascorbic acid.

Acknowledgments

The authors thank to Prof. Dr. Ayşen Gargili Keles, the head of Basic Health Sciences Laboratory which all experiment was conducted in. The present study had some data from the master thesis of Sinem Bahar supervised by Burcu Irem Omurtag Korkmaz.

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Author notes

irem.omurtag@marmara.edu.tr