Establishing Relationship between Vitamins, Total Phenolic and Total Flavonoid Content and Antioxidant Activities in Various Honey Types

Honey is a well-known natural sweetener and is rich in natural antioxidants that prevent the occurrence of oxidative stress, which is responsible for many human diseases. Some of the biochemical compounds in honey that contribute to this property are vitamins and phenolic compounds such as phenolic acids and flavonoids. However, the extent to which these molecules contribute towards the antioxidant capacity in vitro is inconsistently reported, especially with the different analytical methods used, as well as other extrinsic factors that influence these molecules’ availability. Therefore, by reviewing recently published works correlating the vitamin, total phenolic, and flavonoid content in honey with its antioxidant activities in vitro, this paper will establish a relationship between these parameters. Based on the literature, vitamins do not contribute to honey’s antioxidant capacity; however, the content of phenolic acids and flavonoids has an impact on honey’s antioxidant activity.


Introduction
Honey is a natural sweetener that is well-known all around the world. Naturally, it contains a concentrated sugar solution which is mostly fructose and glucose, making it favorable as a substitute for table sugar. It also contains different minor compounds, including polyphenols, enzymes, organic acids, and water-soluble vitamins [1], which contribute to its wide range of biological effects [2]. One of its sought after properties is its ability to counter oxidative stress, thereby acting as a potent antioxidant source.
Oxidative stress causes various pathological conditions such as cancer, neurological disorders, hypertension, and diabetes [3]. However, with a substantial amount of antioxidants the damage can be prevented. An antioxidant is any material that delays or stops the oxidation process of an oxidizable substance [4]. They include both enzymatic (superoxide dismutase, catalase etc.) and non-enzymatic molecules, which are the subject of interest in this review. An example of the important antioxidant compounds in honey are the phenolic compounds such as phenolic acid, flavonoids, and also vitamins [5][6][7].
There are various types of tests that can be used to measure honey's antioxidant capacity in vitro, but none has been declared as the official method. Owing to some modifications

Vitamin and Antioxidant Activity
Vitamins are a large group of complex organic compounds that help to support body metabolism, growth, and development, and regulate the function of cells. To ensure a proper metabolic and cellular reaction in the body, vitamins are required in small amounts to work as important coenzymes and cofactors.
Vitamin A, vitamin C, and vitamin E are known to exhibit certain antioxidant properties. For instance, vitamin C's antioxidant effects are due to its ability to reduce oxidation by reacting with superoxide ion O 2 − and singlet oxygen such as HOO − or OH − through dehydrogenation to generate dehydroascorbate. Vitamin E can remove O 2 − and quench singlet oxygen and superoxide dismutase by working glutathione peroxidase so that consumers have an antioxidant effect inside their body [13]. However, because of honey's nature as an aqueous-based foodstuff [11], most studies only reported the content of water-soluble vitamins, such as Vitamin C and Vitamin B complex.
There are several methods for quantifying vitamins in honey, such as using a fluorometric method, titrimetric method, and chromatograpy [14]. The titrimetric method is widely employed for its simplicity and cost efficiency. However, when considering accuracy and precision, chromatography techniques such as high performance liquid chromatography (HPLC) are preferred [11].
Vitamin C and Vitamin B1, B2, and B3 were detected in honey samples of different botanical origins and geographical locations at various concentrations (Table 1). Manuka honey had the highest level of vitamins (1067.37 mg vitamin C/kg honey), followed by thyme honey (759 mg vitamin C/kg honey). The honey samples that had the lowest vitamin levels were eucalyptus 1 and multifloral honey from Province Leon, both having the same value at 3.40 mg vitamin C/kg honey.
There are numerous reports on vitamins in honey, but only a few studies have statistically analyzed the relationship between vitamin content and antioxidant activity. For example, Chua et al. [11] found that the water-soluble vitamin concentration in Gelam, Tualang, and Acacia honey in Malaysia was significantly correlated with FRAP values (r = 0.4338, p < 0.05). Although there was a high correlation with DPPH activity, it was insignificant (r = 0.8226, p > 0.05). Gelam had the highest vitamin content and the highest antioxidant capacity.
Moreover, Combarros-Fuertes and Azza et al. [15,16] found no significant relationship between vitamin content and DPPH activity in various honey samples. This suggests that vitamins, in small amounts, do not contribute towards the antioxidant activity of a sample. A higher vitamin content has been shown to reduce the β-carotene bleaching inhibition in honey (r = −0.61; p < 0.05), which is believed to be due to the formation of ascorbyl radical [15].

Total Phenolic Content and Antioxidant Activity
Phenolic compounds are one of the most important compounds contributing to the antioxidant activity of honey [5]. Based on carbon chain classification, there are 16 classes of phenolic compounds [31]. In honey, the two most common are phenolic acid (nonflovonoid) and flavonoids [32]. Phenolic content in honey varies among honey types and geographical origins.
The recovery of phenolic compounds is primarily affected by sample preparation and the method of extraction. Some important variables need to be taken into account such as type of solvent and the time and temperature parameters of the extraction process [33]. The most common method used to quantify total phenolic content are the Folin-Denis and Folin-Ciocalteu methods [33]. To identify and quantify individual compounds, chromatography approaches such as HPLC or liquid chromatography (LC coupled with UV-VIS or diode array detector) were adopted [20,[34][35][36][37].
In Table 2, 16 different phenolic compounds detected in honey samples are shown. Overall, multifloral 1 honey from Brazil [34] had the highest phenolic content (78.2 ± 2.7 mg/g GAE), while the lowest was reported in Clover honey (0.65 ± 0.42 mg/g GAE) [35]. Reports have mostly shown a strong correlation between total phenolic content and the antioxidant capacity of honey samples (Table 1).   Lianda et al. [34] investigated Brazilian honey and found multifloral honey to have the highest TPC value (78.2 ± 2.7 mg/g GAE) and the lowest IC 50 value (10.81 ± 0.50 mg/mL). IC 50 corresponds to the sample concentration needed to scavenge 50% of DPPH· radicals. Therefore, low IC 50 is equivalent to a high scavenging power. However, based on FRAP assay, orange blossom 1 honey had the highest FRAP value (438.69 ± 2.78 mol Fe(II)/100 g). Nevertheless, analyses showed a strong correlation between TPC and DPPH IC50 (r = −0.8918) and FRAP assay (r = 0.9258).
In a study by Can et al. [35], the total phenolic content of Turkish honey was strongly correlated with a FRAP assay (r = 0.81, p < 0.05). Chestnut and oak honey had the highest FRAP values, followed by heather, pine, and Jerusalem tea honey.
Similarly, Nascimento et al. [36] also found a strong correlation between TPC and DPPH IC50 activity (r = −0.7582) and FRAP assay (r = 0.8594) in Brazilian honey. In addition, they found abundant gallic acid, a phenolic compound with antioxidant properties in Japanese grape, mastic, and wildflower honey that influenced the FRAP values (r = 0.5202).
Khalil et al. [37] compared Malaysian honey with Manuka honey from New Zealand and discovered that TPC was highly correlated with DPPH % inhibition (r = 0.976, p < 0.01) and FRAP assay (r = 0.965, p < 0.01), showing Tualang honey as the richest in phenolic compounds and highest in radical scavenging activities. The strong correlation of TPC in both assays was also observed in all samples of Kosovo honey [38]. Honey samples from the forest had the highest TPC, DPPH (%), and FRAP values and the correlation with these two assays were (DPPH; r = 0.931, p < 0.01) and (FRAP; r = 0.878, p < 0.01).
However, not all studies showed a strong relationship with the assays. For example, Šarić et al. [39] only discovered a strong correlation in TPC of multifloral honey from Croatia with the FRAP value (r = 0.8325). While the relationship with DPPH IC50 was unfavorable (r = 0.5791) and insignificant. Ranneh [20] also found no significant correlation between TPC and DPPH and ABTS parameters in Tualang and Kelulut honey.
Despite this, most results suggest that phenolic compounds are partially responsible for honey's antioxidant properties.

Flavonoid Content and Antioxidant Activity
Flavonoids are known to be polyphenolic compounds comprising two phenyl rings linked by a propane bridge, resulting in a characteristic 15-carbon (C6-C3-C6) flavan skeleton [40]. They can be regarded as a class of phenolic compounds having a low molecular weight and are widely distributed in the plant kingdom. In higher plants, they represent one of the most distinctive compound groups. In most angiosperm families, nearly all flavonoids are easily detected as flower pigments. Flavonoids can be found naturally in all parts of plants and were discovered to be the major colouring component of the flowering plants [41]. Anthocyanins, flavonols, flavan-3-ols, flavanones, flavones, and isoflavones are some of the main groups of flavonoids.
Honey can be distinguished by its composition, such as the presence of flavonoid compounds. In various plant species, flavonoids are the dominant class of secondary metabolites and occur in various tissues and organs [42]. Antioxidant properties are also controlled by the subgroup of flavonoid compounds found in honey. Each subgroup has different degrees of unsaturation and oxidation of the carbon ring, depending on the location of the C ring attached to the B-ring [43]. The flavonoid subgroups found in honey are mostly flavonols, flavanone, and flavones.
To estimate the flavonoid content in honey, the calorimetric method using aluminium chloride is widely applied [11,20,36,37,39,44,45]. There may be slight modifications from the original procedure but the underlying principle is the same. Flavonoid reacts with aluminium chloride to form a stable acid complex which is detected using spectrophotometer [46]. Then, flavonoids are individually identified and quantified using a chromatographic approach, such as UPLC [11], HPLC [36,37,44], or LC [20]. Table 3 shows the flavonoid content with antioxidant activities in various types of honey. The most common flavonoids detected from different honey samples were gallic acid and caffeic acid. Similarly to TPC, the relationship between TFC and the antioxidant assays mostly showed a moderate to strong correlation. For example, based on DPPH % radical scavenging activity (RSA), there was strong relationship between TFC and DPPH (% RSA). Meanwhile, the lowest (r = 0.888, p < 0.001) was recorded by Khalil et al. [37].  Sousa et al. [47] found a stronger relationship between these two variables in Brazilian honey (r = 0.9377, p < 0.01). Monofloral honey from Jandaira, Brazil, had the highest RSA (46.9 ± 1.9% RSA), with a TFC content of 4.2 ± 0.6 mg GAE/100 g.
A-Farsi [45] investigated Omani honey and found a TFC relationship with DPPH IC50 at r = −0.616. However, Saric et al. [39] observed contradictory results when using two different assays. The correlation between TFC and FRAP assays was higher (r = 0.7062), signaling a flavonoid compound attribution towards the FRAP analysis. However, when evaluating TFC and DPPH IC50 , the relationship was positive, with a relationship coefficient of 0.4272.
Nascimento et al. [36] stated that there was a relationship between TFC and DPPH IC50 , without stating whether it was positive or negative. However, the authors reported a strong relationship between TFC and FRAP values (r = 0.8435, p < 0.005), with Mastic honey being quantified with the highest values of both of these variables.
In the case of Ranneh et al. [20], they observed a significant relationship between TFC of Kelulut (Trigona) and Tualang (Apis dorsata) honey with DPPH % (r = 0.922 and r = 0.936, respectively, p < 0.01), but not with ABTS assay. Kelulut honey had the highest TFC, with 101.5 ± 11.4 mgCE/kg. Overall, the authors concluded that Kelulut honey has a stronger antioxidant capacity than Tualang honey.

Conclusions
This review paper has established correlations between vitamin, total phenolic, and flavonoid contents with the antioxidant activities in vitro. Data on the relationship between vitamins and antioxidant activities are scarce, but based on the available reports, vitamins do not contribute to honey's antioxidant capacity. Although vitamins are well-known antioxidant molecules, their presence in minute amounts could not provide a substan-tial contribution towards honey's antioxidant activity. On the other hand, total phenolic and flavonoid contents are associated with antioxidant activities in vitro. The phenolic compounds are influenced by several factors, including geographical location, botanical origin, type of phenolic compounds, storage duration, and processing method. In addition, because there is no standardized method for measuring honey's antioxidant activity, comparisons between studies are difficult, especially when the expressed units are different (i.e., TPC content and FRAP values). Thus, it is recommended to establish a standard to measure honey's antioxidant capacity, in order to obtain reliable data that can be compared across various studies. This review focused on in vitro antioxidant studies. Naturally, there are limitations to these results, as it does not consider the physiological parameters that can be observed in in vivo settings. The bioavailability and the synergistic/antagonistic effect between these compounds in humans are still vague. However, the strong correlation seen in this review provides fundamental information that phenolic compounds in honey do have positive effects on antioxidant activity. The in vitro studies are increasing rapidly and the in vivo studies are also catching up. More clinical research should instead be done to validate honey as an alternative medicine based on its antioxidant potential.

Conflicts of Interest:
The authors declare no conflict of interest.