Total Antioxidant, Phenolic Compounds, and Total Oxidant Status of Certified and Uncertified Turkey's Honeys

Abstract

In this study, total oxidant status (TOS), total antioxidant capacity (TAC) and total phenols (TP) of seventeen certified and eight uncertified honey samples were analyzed. The TAC and TOS of the samples were determined using novel methods. Uncertified honey samples had significantly lower values for their TP, TAC and TOS contents than those of certified honey samples. There was significant correlation between TOS and TP values (P < 0.01) and between TOS and TAC values (P < 0.05 ).

Keywords:

• Honey
• Total antioxidant capacity
• Total oxidant status
• Total phenols

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Introduction

Honey is a natural substance produced by honeybees, Apis mellifera, from flower nectar or from honeydew. It is a very important energy source food (0.07 J/100 g honey) being a rich source of carbohydrates mainly fructose (about 38.5 g/100 g sample) and glucose (about 31.0 g/100 g sample) and it also contains a very complex mixture of other saccharides, enzymes, amino and organic acids, polyphenols, carotenoid-like substances, Maillard reaction products, vitamins, and minerals.[1]

Recently, functional foods have received attention. Antioxidants play an important role for human health by combating damage caused by oxidizing agents and for food preservation[2]where they specifically retard deterioration, rancidity or discoloration due to oxidation caused by light, heat and some metals. Development of oxidative stress, which has been implicated in over 100 disorders,[3 ,4] can be removed by enzymatic and nonenzymatic antioxidative mechanisms in organisms. Food antioxidants include both enzymatic (e.g., catalase and glucose oxidase) and non-enzymatic substances (e.g., organic acids, Maillard reaction products, amino acids, proteins, flavonoids, phenolics, α-tocopherol, flavonols, catechins, ascorbic acid, and carotenoids).[1]

Honey, naturally contains a number of components known as antioxidants including chrysin, pinobanksin, vitamin C, catalase and pinocembrin[1] and phenolic compounds.[5] Honey was found to be effective in increasing antioxidant agents, trace elements, serum iron and blood indices, and in decreasing fasting blood sugar.[6]

The use of honey in the treatment of chronic wounds and diabetic ulcers, cataracts, and other eye ailments and peptic ulcers and other gastric ulcers has been documented. This therapeutic role of honey was partially attributed to its antibacterial activity.[7] The antibacterial activity of honey is attributed due to: acidity, low osmolarity and chemical compounds (such as hydrogen peroxide, volatiles, beeswax, nectar, pollen and propolis).[8,9] White et al.[10] reported that the major antibacterial factor in honey was due to hydrogen peroxide, which is produced by glucose oxidase originating from hypopharyngeal glands of honey bees. Catalase, which originates from pollen, also founds in honey. The level of hydrogen peroxide in a given honey is determined by relative levels of glucose oxidase and catalase.[8] The higher the glucose oxidase level, the higher the peroxide content, whilst the higher the catalase content, the lower the peroxide level. Differences in antimicrobial activity among honeys from various floral sources may be, in part, a reflection of these variations.[11] Total oxidant status measurements can give an idea about the level of hydrogen peroxide contents of the honeys because it is the major oxidative molecule in honeys.

The importance of honey has been recently upgraded because of its nutrient and therapeutic effects. Since the adulteration of honey has been seen as prevalent, the need has arisen for more effective and simpler quality control methods for detecting adulteration. The authenticity of honey has two different aspects: authenticity with respect to honey production and authenticity with respect to description. The determination of honey authenticity requires the consideration of both aspects. There appears to be limited data for the total phenolic contents, antioxidant activities and total oxidant contents of Turkish honeys. The aim of this study was to evaluate and to compare the total oxidant status, total antioxidant capacity and phenol contents of certified and uncertified honey samples with a discussion of their possible role in the detection of honey authenticity.

MATERIALS AND METHODS

Honey Samples

Seventeen most common certified Turkish honeys with a guarantee of genuineness and known history and eight uncertified honey samples with unknown history were obtained from specific honey producers and non-specific markets. Uncertified honey samples were assessed as adulterated in production by the honey experts. The names of the samples were given with respect to their origins and common names and the uncertified ones were given numbers. All the samples were stored in a dark room at 20°C before the samples were used for tests. Triplicate analysis was conducted within two months after collection.

Chemicals and Apparatus

All chemicals were ultra pure grade (Sigma Co. and Merck Co.) and type I reagent grade deionized water was used. A Cecil 3000 spectrophotometer with a temperature controlled cuvette holder (Cecil), and an Aeroset automated analyzer (Abbott) were used.

Total Antioxidant Capacity (TAC) Determination

TAC values of the samples were determined according to a method described by Erel.[12] The samples were added to pure methanol at a ratio of 1/1 (v/v) and tested for TAC, total oxidant status (TOS) and total phenol. Total antioxidant capacity values (TAC) of the samples were expressed as mmol Trolox equivalent/L.

Total Oxidant Status (TOS) Determination

TOS levels of the samples were determined according to Erel's method.[13] The assay is based on the oxidation of ferrous ion to ferric ion in the presence of various oxidant species in acidic medium and the measurement of the ferric ion by xylenol orange. In this method, oxidants oxidize ferrous-o-dianisidine complex to ferric ion. The produced ferric ion makes a colored xylenol orange-ferric ion complex. The color intensity was related with the oxidant amount of the sample. The method was applied to an automated analyzer, which was calibrated with hydrogen peroxide. The values are given as μmol H₂O₂ equivalent/L of honey sample.

Total Phenol Content Determination

Total phenol contents of the samples were determined using Folin–Ciocalteu reagent described by Skerget et al.[14] Prepared sample was measured at 760 nm against a methanol blank. Gallic acid (0–200 mg/l) was used as standard to produce the calibration curve. The total phenolic content was expressed in mmol gallic acid equivalent/L of honey.

Statistical Analysis

Results are presented as mean values. Data were tested using SPSS for Windows Release 10 (SPSS Inc.). Statistical analysis of the results was based on Mann-Whitney U test and correlation analyses. Statistically significant differences were considered at the level of P < 0.05 unless otherwise given.

RESULTS AND DISCUSSION

TAC, TP and TOS results of the samples are presented in Tables 1 and 2. The mean values (Table 2) were significiantly higher for the certified honey samples than the uncertified ones. The mean TAC values of certified and uncertified samples were 0.216 and 0.087 mmol Trolox equivalent/L and the mean TP contents of them were 17.144 and 10.930 mmol gallic acid equivalent/L, respectively. The average content of total phenols was in good agreement with that reported in literature.[15,16] The total phenolic contents of certified honey samples were within the same range as the literature data of Turkish chestnut honeys,[17] at the higher limits of some literature values[7,15,18] and at the lower limits of some literature values[17] when the modifications of the methods were taken into account. The concentration and type of phenolic substances depend on the floral origin of honey. Blasa et al.[19] suggest that the unprocessed, darker, opaque honeys contained the highest levels of total phenolics.

Table 1 Total antioxidant capacity (TAC), Total phenol (TP), and Total Oxidant Status (TOS) contents of honey samples (n:3)

No	Name	Group^b	TAC^a	T. Phenol^a	TOS^a
1	Elmadag Floral (Type 1)	C	0.1830 ± 0.0021	18.92 ± 0.30	12.83 ± 0.23
2	Elmadag Floral (Type 2)	C	0.2376 ± 0.0054	16.58 ± 0.15	13.43 ± 0.18
3	Adana Ucgul	C	0.1204 ± 0.0023	17.02 ± 0.16	6.68 ± 0.12
4	Natural Old Honey	C	0.2541 ± 0.0029	18.39 ± 0.25	9.42 ± 0.10
5	Aydın Pine	C	0.5131 ± 0.0022	16.71 ± 0.21	11.21 ± 0.22
6	Bitlis Floral	C	0.0490 ± 0.0009	16.73 ± 0.17	4.60 ± 0.31
7	Polatlı Floral	C	0.2663 ± 0.0057	16.37 ± 0.18	9.58 ± 0.11
8	Kirikkale Floral	C	0.1604 ± 0.0010	16.82 ± 0.12	7.30 ± 0.09
9	Diyarbakir Karacadag Geven	C	0.0971 ± 0.0008	16.75 ± 0.40	6.05 ± 0.23
10	Malatya Floral	C	0.0908 ± 0.0013	19.85 ± 0.29	5.04 ± 0.12
11	Sandal-Citrus mixed	C	0.2090 ± 0.0022	16.78 ± 0.36	4.12 ± 0.03
12	Kayseri Tomarza Floral-thyme	C	0.5755 ± 0.0102	16.40 ± 0.12	9.24 ± 0.08
13	Cotton-Floral mixed 2	C	0.1495 ± 0.0011	16.80 ± 0.31	17.99 ± 0.33
14	Diyarbakir Floral	C	0.1769 ± 0.0010	16.83 ± 0.22	10.97 ± 0.14
15	Elazıg Geven	C	0.0871 ± 0.0005	16.68 ± 0.51	8.29 ± 0.09
16	Aydın Floral	C	0.2762 ± 0.0041	16.89 ± 0.46	2.79 ± 0.04
17	Muş Bulanık Floral	C	0.2269 ± 0.0019	16.93 ± 0.35	6.41 ± 0.04
18	Honey No:1	U	0.0857 ± 0.0010	11.84 ± 0.18	2.50 ± 0.02
19	Honey No:2	U	0.1320 ± 0.0020	11.00 ± 0.12	1.20 ± 0.04
20	Honey No:3	U	0.0854 ± 0.0009	9.61 ± 0.05	1.40 ± 0.02
21	Honey No:4	U	0.2155 ± 0.0044	9.45 ± 0.09	0.43 ± 0.01
22	Honey No:5	U	0.0585 ± 0.0010	9.14 ± 0.10	0.70 ± 0.05
23	Honey No:6	U	0.0767 ± 0.0016	13.74 ± 0.32	0.90 ± 0.01
24	Honey No:7	U	0.0223 ± 0.0008	12.37 ± 0.39	1.49 ± 0.03
25	Honey No:8	U	0.0165 ± 0.0013	10.29 ± 0.20	0.53 ± 0.04
^aUnit of TAC: mmol Trolox equivalent/L; unit of total phenol: mmol gallic acid equivalent/L, unit of total oxidant status: μmol H2O2 equivalent/L; ^bC: certified; and U: uncertified.

Table 2 Descriptives of the honey samples for their total antioxidant capacity (TAC), total phenol (TP), and total oxidant status (TOS) contents. (P < 0,05)

	Type	N	Mean	Std. Deviation	Std. Error
TAC*	Uncertified	8	0.087	0.064	0.023
	Certified	17	0.216	0.141	0.034
	Total	25	0.175	0.135	0.027
TP*	Uncertified	8	10.930	1.619	0.572
	Certified	17	17.144	0.962	0.233
	Total	25	15.156	3.184	0.637
TOS*	Uncertified	8	1.144	0.673	0.238
	Certified	17	8.585	3.898	0.945
	Total	25	6.204	4.777	0.955
^aUnit of TAC: mmol Trolox equivalent/L; unit of total phenol: mmol gallic acid equivalent/L; unit of total oxidant status: μmol H2O2 equivalent/L.

According to the results, the certified honey samples had the total oxidant status values changing between 2.79 to 17.99 μmol H₂O₂ equivalent/L (Table 1) and the mean total oxidant status of the uncertified honey samples (1.144 μmol H₂O₂ equivalent/L) were significiantly (p = 0.05) lower than that of the certified honey samples (8.585 μmol H₂O₂ equivalent/L).

The correlation analysis (Table 3) showed that, there were significant positive correlations between TAC and TOS (P < 0.05) of the honeys and TP and TOS (P < 0.01) of the honeys. The existence of both of the antioxidants and oxidants as positively correlated in honey makes it evident that there are mechanisms in honey controlling the balance of reactive oxygen species.

Table 3 Correlation analysis of the honey samples for their total antioxidant capacity (TAC), total phenol (TP), and total oxidant status (TOS) contents

		TAC^a	T. Phenol^a	TOS^a
TAC^a	Pearson Correlation	1.000	0.348	0.444*
	Sig. (2-tailed)	–	0.89	0.026
TP^a	Pearson Correlation	0.348	1.000	0.689**
	Sig. (2-tailed)	0.089	–	0.000
TOS^a	Pearson Correlation Z	0.444*	0.689**	1.000
	Sig. (2-tailed)	0.026	0.000	–
*Correlation is significant at the P = 0.05 level; and**correlation is significant at the P = 0.01 level. ^aUnit of TAC: mmol Trolox equivalent/L, Unit of total phenol: mmol gallic acid equivalent/L, unit of total oxidant status: μmol H2O2 equivalent/L.

The discrepancies in the antioxidant values of certified honey samples could be attributed to the differences of botanical sources of honey and also to the presence of different compounds of antioxidants. Beretta et al.[15] found that floral origin was the major determinant in the antioxidant potency of natural honeys. In literature, when in vivo antioxidant effect of honey was studied; serum antioxidant capacity increased significantly (p < 0.05) following consumption of buckwheat honey in water and phenolic antioxidants of processed honey were found as bioavailable, and they increase antioxidant activity of.[20,21] Gheldof et al.[22] analyzed honeys from seven different floral sources for in vitro antioxidant capacity and total phenolic content. Antioxidant capacity measured by the oxygen radical absorbance capacity (ORAC) assay ranged from 3.1–16.3 mmol Trolox equivalent/L honey. A linear correlation was observed between phenolic content and ORAC activity of the investigated honeys (p < 0.0001, R (2) = 0.9497) showing that honey may be used as a healthy alternative to sugar in many products and thereby serve as an enhanced antioxidant defense system in healthy adults.

Many types of honey are produced in Turkey although detailed investigations on their chemical and biological properties are very limited.[23] Anklam[24] reported that there is no direct parameter measured to determine the exact quality and biological activity of honey and that the routine chemical tests performed do not provide dependable information about the quality of honey. According to Lachman et al.[25] the combination of methods based on multi-component analysis with the support of statistical data evaluation seems to be a useful approach for determining the authenticity of honeys. The current study was designed to assess the total antioxidant, phenol and total oxidant status of certified and uncertified honey samples from Turkey and investigate the contribution of these data on determining authenticity of honey.

Many of the flavonoids and phenolic acids identified have been previously described as potent antioxidants. In a study carried out by Gheldof and Engeseth,[26] chromatograms of the phenolic nonpolar fraction of various honey samples showed similar but quantitatively different phenolic profiles. A positive correlation was observed between percentage antioxidant capacity and total phenolics content by Al-Mamary et al.[18] In general, the antioxidant capacity of honey appeared to be a result of the combined activity of a wide range of compounds including phenolics, peptides, organic acids, enzymes, Maillard reaction products, and possibly other minor components. The phenolic compounds contributed significantly to the antioxidant capacity of honey but were not solely responsible for it.[22] Moreover, Beretta et al.[15] stated that the total phenolic content of natural samples, such as plants and honey, reflected, to some extent, total antioxidant capacity of samples; so the presence of constituents such as vitamin C, E, and carotenoids may influence the total antioxidant activity.[27] According to the results of this study, there was not a significant correlation between TAC and TP contents of certified honey samples (Table 3) suggesting that the novel TAC method used in this study measures not only the phenolic antioxidants but also the other compounds having antioxidant property.

The absolute level of hydrogen peroxide in any honey was said to be determined by the respective levels of glucose oxidase and catalase enzymes in that honey. The higher the glucose oxidase level, the higher the peroxide level and the lower the catalase level, the higher the peroxide level.[28] As the glucose oxidase in honey is known to be originated from bees, a similar glucose oxidase level in most honeys world-wide might be expected. On the other hand, because catalase originates in plants, the level of catalase in honey will effectively determine the level of peroxide in a honey. Hydrogen peroxide that most of the antimicrobial activity of honey has been attributed to, is produced by naturally occurring glucose oxidase. The presence of hydrogen peroxide, as well as some minerals (particularly copper and iron), in honey may lead to the generation of highly reactive hydroxyl radicals as part of the antibacterial and also oxidant system.29–31

There is no official method for antioxidant determination of honey but various methods as the DPPH (1,1-diphenyl-2-picrylhydrazyl) method, FRAP assay (ferric reducing antioxidant power), ORAC (oxygen radical absorbance capacity), superoxide radical-scavenging activity, and TEAC (Trolox equivalent antioxidant activity) are in use. In this study, the easy, inexpensive and fully automated methods described were used to measure TAC and TOS of samples. Because plant foods contain many different classes and types of antioxidants and interactions among them can affect total antioxidant capacity, producing synergistic or antagonistic effects,[32] knowledge of their total antioxidant capacity (TAC), which is the cumulative capacity of food components to scavenge free radicals, would be useful for epidemiologic purposes.[33]

Honey authenticity is a complex problem, which currently has a significant economic impact and undeniable nutritional and organoleptic consequences. Though, there are a lot of methods for evaluation of indicatives of honey quality, various methods, in combination with multivariate statistical techniques could be used for the detection of the authenticity and botanical origin of honey. A common feature of the various regulations is the specification of standards that set the upper or lower limits for the levels of water, reducing sugars, sucrose, minerals, and hydroxymethylfurfural (HMF) in honey. These specifications serve, to some degree, to exclude honeys subjected to some of the common practices of adulteration.[34] Because, for example to detect the addition of the high fructose syrups and invert sugars, HMF test is used. However, HMF can be legally present in honeys in quite high amounts depending on the origin of honey besides other factors.[35] It is stated by Guler et al.[36] that the sugar (sucrose, fructose and glucose) content of honey cannot be used to distinguish between adulterated (sucrose syrup) and pure blossom honey. The characteristics and composition of honey can also vary considerably according to the honey type and the geographical or botanical origin and the sugar content of honey quantitatively and qualitatively exhibits a distribution of values rather than a unique value. Moreover, some of the experimental parameters may be arranged later on honey by some manipulations by adding some chemicals. Therefore, it is very difficult to decide that there is authenticity by a single method. The results in this study indicated that TAC and TP values, and TOS results may be potent criteria to identify authenticity of honey.

CONCLUSION

The mean TAC, TP, and TOS values of the certified honey samples were significiantly higher than those of the uncertified ones suggesting that these parameters may be used as potent criteria for the identification of authenticity in honey. According to the results, the TOS values were found to be significantly and positively correlated both to the antioxidants and oxidants indicating that there are mechanisms in honey controlling the balance of reactive oxygen species. For further studies this method requires a detailed database of the natural variations expected for each variety of honey in order to be useful in detecting adulteration.