Honey is a complex mixture, containing ~180 compounds, produced by the
Honey and other bee products contain plant antioxidants that have high bioactivity levels and chemical diversity (
The western honeybee,
Honey is a complex mixture, which consists of a variety of ~180 compounds, such as carbohydrates, mainly including glucose and fructose (60–85%), water and minority compounds, such us phenolic compounds, minerals, proteins, enzymes, free amino acids and vitamins (
Some of the minor constituents of honey, compared to its major sugar levels, are considered to have antioxidant properties (
Honey may emanate both from single (unifloral honey) or multiple (multifloral honey) plant species depending on the diet of bees (
According to previous research, honey may represent an anticancer agent (
There are a few studies investigating the antitumor effects of honey on liver cancer cells, demonstrating that honey is able to reduce the levels of nitric oxide (NO) in the cells and decrease the HepG2 population as well, improving the total antioxidant profile of the cells (
Honey samples were collected from two different regions in Greece from small-scale producers. Specifically, the first region was Taygetos mountain in Peloponnisos and from different areas of the longest mountain range in Greece, particularly from Pindos. For the purpose of the study, blind sampling was used. The only data available were the type of honey, the beehive location and the harvest date. In total, six different types of raw honey were collected (
The estimation of TPC of the different types of honey was performed by the use of Folin-Ciocalteu (FC) reagent. A total of 20 µl of each sample, 1 ml dH2O and 100 µl FC reagent were added to test tubes, followed by incubation for 3 min in room temperature (RT), in the dark. Subsequently, 25%w/v of sodium carbonate solution (280 µl) and 600 µl dH2O were added followed by incubation for 1 h at RT and under dark conditions. A test tube including only FC and dH2O was used as a blank and the absorbance was measured at 765 nm using a spectrophotometer (Hitachi, U 1900 UV/VIS, Hitachi High-Technologies Corporation). For the estimation of TPC, a gallic acid standard curve was made (at concentrations of 0, 50, 150, 250 and 500 µg/ml) and later used for the expression of the results as/mg of gallic acid equivalents (GAEs) per g of honey (mg GAE/g honey) (
The free-radical scavenging capacity (RSC) of the honey samples was evaluated using an assay originally described by Brand-Williams
where ODcontrol is the absorbance value of the control solution, and ODsample is the absorbance value of the sample. Through the graph-plotted RSC percentage against the honey concentration, the half maximal inhibitory concentration (IC50) was calculated to compare the inhibition of radical capacity of the honey samples. The experiment was conducted at least three times independently.
A slightly modified version of the original one was used to evaluate the ability of the honey samples to inhibit radicals (
An altered version of the one described by Chung
For the evaluation of the superoxide anion radical-scavenging ability of the honey, an altered version of the method described by Gülçin
The assay was performed with some modifications, as previously described by Tekos
In the equation, S represents the percentage of the supercoiled plasmid DNA in the tested samples, S° refers to the percentage of the supercoiled plasmid DNA in the positive control, and Scontrol represents the percentage of the supercoiled DNA in the negative control. The IC50 value was determined as described above. The experiment was conducted at least three times independently.
The reducing power assay was performed as previously described by Yen and Duh (
The HepG2 cell line was donated by Assistant Professor Kalliopi Liadaki (Department of Biochemistry and Biotechnology, University of Thessaly, Larissa, Greece). HepG2 cells are frequently used to examine the effects of unidentified compounds with potential anticancer activity, as they maintain the activities of numerous enzymes crucial for xenobiotic metabolisms. When studying complex matrices such as honey, which contains a variety of biologically active chemicals and whose efficacy may fluctuate due to metabolic transformation, the selection of the cell line is crucial (
The cells were cultured in normal Dulbecco's modified Eagle's medium (DMEM), containing 10% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml of penicillin and 100 U/ml of streptomycin (all from Gibco; Thermo Fisher Scientific, Inc.; complete medium) in plastic disposable tissue culture flasks at 37°C in 5% carbon dioxide. The experiment was conducted at least three times independently.
Cell proliferation was examined using the XTT assay kit (R&D Systems, Inc.). A total of 10,000 cells were placed in each well of a 96-well plate in complete DMEM and incubated for 24 h at 37°C. The cells later treated with a range of concentrations of raw honey in DMEM in the absence of FBS. After 24 h, 50 µl of the XTT solution, containing 49 µl XTT-labeling reagent with 1 µl XTT activator were added to each well of the plate followed by incubation for 4 h at 37°C and the measurement of the optical density at 450 and 630 nm (which is the reference wavelength) using a plate reader (Bio-Tek ELx800; Bio-Tek Instruments, Inc.). The absorbance of each tested concentration of each tested honey was measured without cells, as well as cell cultures in the absence of sample (control), and in the absence of cells (negative control), using the same plate reader. The absorbance values obtained in wells that contained only raw honey samples were subtracted from those acquired from wells that contained the respective extract concentration and seeded cells. Data were calculated as follows: % (of control) cell viability=(ABsampe/ABcontrol) ×100, where ABcontrol and ABsample indicate the optical density of the negative control and the test compounds, respectively. All experiments were carried out in triplicate and at least on two separate occasions.
For the purpose of measuring the GSH and ROS levels, the HepG2 cells were cultured in a six-well plate and incubated for 24 h at 37°C, 5% CO2 and 80–95% humidity in complete medium, until they reached a confluency of 70–80%. On the following day the complete medium was replaced with serum-free medium with the following honey concentrations: 3.125-25 mg/ml of oak, EC, FOH, F1 and 1.56-12.5 mg/ml of FV and F2, and incubated for 24 h at 37°C. In order to measure the GSH levels, the cells were collected by trypsinization and washed with PBS twice following consecutive centrifugations at 300 × g for 5 min at 4°C. Following each centrifugation step, the supernatant was discarded, and the cellular pellet (106 cells/ml) was resuspended in PBS. Following the second wash, the cells were incubated in 1 ml PBS, including 5 µl Thiol Green dye (Thiol Green Indicator, Abcam), at 37°C for 30 min under dark conditions with the obligation of slightly mixing every 10 min under dark conditions followed by centrifugation (300 × g, 5 min, 4°C) and resuspension in PBS. Thiol Green accumulates primarily in the cytosol in normal cells; however, when the cells are apoptotic, it is able to partially translocate to the mitochondria, while its staining intensity is decreased (
For the determination of the levels of total antioxidant capacity (TAC), TBA reactive substances (TBARS) and protein carbonyls (PCARBS), the cells were lysed in PBS with protease inhibitors (Complete™ mini protease inhibitors, Roche Applied Science) at 1×106 cells/ml by sonication. The protein concentration was measured using the Bradford assay and subsequently, a modified method as previously described by Patsoukis
The Janaszewska and Bartosz (
The assay was performed as previously described by Keles
The determination of PCARBS levels was based on the method previously described by Patsoukis
All chemicals used for all the aforementioned assays were supplied by Sigma-Aldrich; Merck KGaA.
For the
The results of the assays performed using
In the DPPH• assay, a wide range of IC50 values was observed. The FOH honey had the highest scavenging activity, followed by the FV and oak honeys. The results of the ABTS•+ assay revealed that the FOH and FV honeys were those with the highest antioxidant capacity. In the hydroxyl radical assay, the flower (F1 and F2) honeys exhibited the lowest IC50 values, which indicates the highest antioxidant capacity, despite the fact that they had the lowest polyphenolic content. The FV and FOH honeys had the highest efficacy in the superoxide radical assay, as shown by their capacity to scavenge efficiently DPPH•, ABTS•+ and superoxide radicals.
The antigenotoxic activity of the raw honey samples was determined by the plasmid relaxation assay, in which the FOH honey displayed the most potent antioxidant capacity as a result of the lowest IC50 value.
The honey samples exhibiting the highest reducing capacity were the oak and FOH honeys. The same samples followed by the FV honey, exhibited the highest polyphenolic content, and the highest ability to inhibit DPPH•, ABTS•+ and superoxide radicals (
As demonstrated by the results presented in
All six of the raw honey samples that were tested in the
The manufacturer's instructions for the XTT assay kit were followed in order to determine which concentration of the samples impede cell growth (i.e., effect on cell viability). The samples were administrated in a liver cancer cell line (HepG2;
In order to examine the effects of the raw honey samples on the HepG2 cells, the highest non-cytotoxic concentrations of each sample were selected. The selected concentrations were used to treat the cells and their effects on the intracellular GSH and ROS levels, as well as on the TAC, TBARS and protein carbonyls levels were assessed.
According to the results obtained using the oak honey, treatment of the cells with lowest concentration (3.125 mg/ml) increased the TAC and lipid peroxidation levels, as compared to the control group. Furthermore, the highest administered concentration (25 mg/ml) increased the GSH, TAC, and TBARS levels in comparison with the control group (
The results regarding the EC honey are presented in
As regards the results of the FV honey (
The results of the FOH honey are presented in
The results of the F1 honey presented in
As regards the results of the F2 honey (
Representative results from the flow cytometry experiment are presented in
The objective of the present study was to determine the antioxidant capacity of honey produced on a small scale throughout Greece by analyzing its effects using cell-free assays, and on a liver cancer cell line (HepG2) redox status using
Scientific research on antioxidants derived from natural products has gained global interest mainly due to their potential positive health effects. Oxidative stress is a condition in which an imbalance between ROS production and antioxidants occurs, leading to cellular damage and the dysregulation of metabolism, associated with several pathological pathologies, such as cancer, cardiovascular diseases, diabetes etc. (
In order to determine the antioxidant capacity of the raw honey samples, the present study performed cell-free and cell-based assays. The TPC levels ranged between 1.32 mg GAE/g for the FV honey to 0.84 mg GAE/g for the EC honey, which was the one with the lowest polyphenol content. These levels reveal differences from previous research, which may be due to the location of the beehives affecting the biodiversity of the geographical region as well (
Subsequently, the samples were tested for their capacity to inhibit DPPH•, ABTS•+, and hydroxyl and superoxide radicals. The FV, oak and FOH honeys were the most effective in terms of their low IC50 values. Honeydew is comprised of secretions of the living parts of plants or the excretions sap-sucking insects; honeybees are able to find this on plants and collect it (
The samples were also examined for their reducing capacity that is strongly associated with their antioxidant capacity. Substitutes with reducing activity are electron donors and are able to reduce lipid peroxidation (
In the plasmid relaxation assay, the results revealed that the FOH and FV honeys were the most effective at protect DNA from single-strand breaks induced by peroxyl radical (ROO•). This assay is used in order to evaluate the antioxidant capacity of a sample as ROO• constitute components of autoxidation and can be easily formed by the decomposition of azo compounds (
The antioxidant capacity of honey is well-established, although the precise mechanisms of action are not yet fully understood (
GSH is the most abundant endogenous antioxidant, playing crucial roles in the detoxification and metabolic processes (
Considering the results for the EC honey, the administration of the highest tested concentration, i.e., 25 mg/ml, disrupted the intracellular redox balance and induced molecular damage, as indicated by the significant increase in TBARS levels. It may be hypothesized that the promotion of lipid peroxidation was responsible for the increased cell death, which however was not statistically significant, as observed in the XTT assay. In the same sample, the lowest concentration used perturbed the redox homeostasis, a finding supported by the elevation in PCARBS levels. The activation of cellular antioxidant mechanisms, as indicated by the significant increase in TAC levels, was not sufficient to prevent oxidative protein damage. As regards the FV honey, the highest concentrations used (6.25 and 12.5 mg/ml) reduced the intracellular ROS levels, while an increase in lipid peroxidation levels was observed at 12.5 mg/ml. Furthermore, the intermediate concentrations (6.25 and 3.125 mg/ml) increased PCARBS in comparison with the control group. Protein carbonylation is widely used as a reliable indicator of oxidative damage (
FOH, a honeydew honey as previously described, is characterized by its dark color and has a different composition due to the plant and insect exudates (
As regards the oak honey, harmful effects were detected at various concentrations. More elaborately, the lowest concentration (3.125 mg/ml) perturbed the redox homeostasis, an assertion supported by the elevated promotion in lipid peroxidation. The activation of antioxidant defenses, expressed through the significant increase in TAC levels, was not sufficient to protect from the induction of molecular damage. Similarly, the highest concentration used (25 mg/ml) disrupted the intracellular redox homeostasis, as indicated by the significant increase in lipid peroxidation levels. The intensification of the cellular antioxidant defenses, evidenced by the significant increase in GSH and TAC levels, was not able to prevent the severe oxidative damage that led to cell death, as confirmed by the cell viability assay. An interesting finding of the present study was the emergence of an hormetic phenomenon in the TBARS levels. More specifically, the elevated levels of lipid peroxidation by-products, which were observed in the lowest concentration used, were followed by the return of TBARS to normal levels, whereas the administration of the highest concentration promoted lipid peroxidation once again. This phenomenon was also observed in the TAC levels. Notably, it has been demonstrated that moderate levels of reactive species cause cell adaptations to stress conditions, a phenomenon known as hormesis. Hormesis is a biological phenomenon that describes the capability of living systems, from a single cell to an organism, to adapt following exposure to low doses or intensity of a stressor (
The six samples in the present study appeared to respond differently to the induction of the biomarkers examined, without exhibiting any specific pattern to the honey concentration used or in the type of biomarker measured. The global literature indicates that honeys with different floral sources have different biochemical profiles (
The limitations of the present study comprise the lack of the evaluation of the bioactive compounds and of the examination of pesticide contaminations in the honeys tested. Nevertheless, in the present study, the aim was to investigate the biological effects of the honey samples as a total mixture, including all bioactive compounds. These biological effects are categorized between the samples tested, without indicating the biological action of specific compounds. The authors aim to conduct measurements concerning the determination of the bioactive compounds of these samples in future research.
According to the scientific literature, exposure to pesticides has a substantial impact on the development and progression of a wide spectrum of chronic diseases in human populations, depending on the levels of environmental exposure. Investigations using laboratory animals designed to evaluate the toxicological profile of pesticide mixtures, administered at concentrations below the existing regulatory limits, have revealed the manifestation of detrimental effects when assessed by metabolomics contrary to the conventional biochemical measurements (
In conclusion, the present study demonstrates that the examined raw honey samples exhibited potent antiradical, reducing and antigenotoxic properties in
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
DK supervised the study and conceived the technical details and designed the experiments. DAS participated in designing the present study and in reviewing the data. AP performed the experiments. AP, ZS and PV analyzed the data. AP and DK confirm the authenticity of all the raw data. AP, PV and ZS wrote the manuscript. All authors have read and approved the final version of the manuscript.
Not applicable.
Not applicable.
DAS is the Editor-in-Chief for the journal, but had no personal involvement in the reviewing process, or any influence in terms of adjudicating on the final decision, for this article. The other authors declare that they have no competing interests.
glutathione
reactive oxygen species
glutathione S-transferase
catalase
malondialdehyde
deionized water
total phenolic content
nitric oxide
folin-ciocalteu
gallic acid equivalents
radical scavenging
horseradish peroxidase
room temperature
trichloroacetic acid
total antioxidant capacity
thiobarbituric acid reactive substances
thiobarbituric acid
2,4-dinitrophenylhydrazine
2,2-diphenyl-1-picrylhydrazyl
2,2-diphenyl-1-picrylhydrazine
peroxyl radical
protein carbonyls
standard error
standard error of the mean
Cytotoxic concentration threshold (mg/ml) in the HepG2 cells, as assessed using XTT assay for (A) oak honey, (B)
Effects of the honeys on GSH, ROS, TAC, TBARS and PCARBS levels in HepG2 cells following 24 h of exposure. (A) Oak honey, and (B)
Effects the honeys on GSH, ROS, TAC, TBARS and PCARBS levels in HepG2 cells following 24 h of exposure. (A) Fir and vanilla honey and (B) forest with honeydew honey. *P<0.05, significant difference compared with untreated HepG2 cells (control). GSH, glutathione; ROS, reactive oxygen species; TAC, total antioxidant capacity; TBARS, thiobarbituric reactive substances; PCARBS, protein carbonyls.
Effects of the honeys on GSH, ROS, TAC, TBARS and PCARBS levels in HepG2 cells following 24 h of exposure. (A) Flower (
Scatter plots and histograms from flow cytometric analysis for the determination of glutathione levels in the HepG2 cell line. The control plots apply to all sample concentrations. The plots are representative of the oak honey sample. (A) Control, (B) concentration of honey at 25 mg/ml, (C) concentration of honey at 12.5 mg/ml, (D) concentration of honey at 6.25 mg/ml, and (E) concentration of honey at 3.125 mg/ml.
Scatter plots and histograms from flow cytometric analysis for the determination of reactive oxygen species levels in the HepG2 cell line. The control plots apply to all sample concentrations. The plots are representative of the oak honey sample. (A) Control, (B) concentration of honey at 25 mg/ml, (C) concentration of honey at 12.5 mg/ml, (D) concentration of honey at 6.25 mg/ml, and (E) concentration of honey at 3.125 mg/ml.
Total phenolic content, and IC50 and AU0,5 values of the raw honey samples obtained using
Honey type | TPC (mg GAE/g) | DPPH• IC50 (mg/ml) | ABTS•+ IC50 (mg/ml) | OH• IC50 (mg/ml) | Superoxide radical IC50 (mg/ml) | Reducing power AU0,5 (mg/ml) | Plasmid relaxation assay IC50 (mg/ml) |
---|---|---|---|---|---|---|---|
Oak | 1.24 | 7.14±0.02 | 2.96±0.81 | 1.22±0.04 | 1.98±0.04 | 1.87±0.19 | 2.98±0.11 |
0.84 | 9.95±0.025 | 4.03±0.08 | 1.04±0.06 | 7.48±0.37 | 3.60±0.3 | 6.04±0.19 | |
Fir and vanilla | 1.32 | 6.51±0.32 | 1.03±0.01 | 1.05±0.06 | 1.01±0.01 | 2.41±0.01 | 1.60±0.17 |
Forest with oak honeydew | 1.16 | 4.61±0.29 | 0.90±0.01 | 1.24±0.02 | 1.24±0.01 | 1.79±0.06 | 1.55±0.15 |
Flower ( |
0.86 | 15.04±0.3 | 1.99±0.1 | 0.68±0.01 | 4.32±0.14 | 3.71±0.25 | 9.02±0.41 |
Flower ( |
0.89 | 8.47±0.69 | 1.45±0.02 | 0.66±0.01 | 2.63±0.02 | 2.28±0.01 | 6.86±0.68 |
TPC, total phenolic content; DPPH•, 2,2-diphenyl-1-picrylhydrazyl; ABTS•+, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid; OH•, hydroxyl radical; IC50, half maximal inhibitory concentration; GAE, gallic acid equivalent.
Correlation coefficient (Rho) values estimated from the correlation analysis between the TPC values and the other
Correlation analyzed | Rho value | P-value |
---|---|---|
TPC vs. DPPH• | −0.771 | P=0.1028 |
TPC vs. ABTS•+ | −0.543 | P=0.2972 |
TPC vs. O2 | −0.943 |
P=0.0167 |
TPC vs. OH• | 0.543 | P=0.2972 |
TPC vs. RP | −0.543 | P=0.2973 |
TPC vs. ROO | −0.657 | P=0.175 |
P<0.05, statistically significant correlation. TPC, total phenolic content; DPPH•, 2,2-diphenyl-1-picrylhydrazyl; ABTS•+, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid; OH•, hydroxyl radical; RP, reducing power; ROO, peroxyl radical.