Antioxidant Profile of Buckwheat Honey from the Republic of Moldova

The honey is a natural substance, whose therapeutic qualities have been developed in parallel with human evolution. Its antioxidant and antiinflammatory properties are due to the polyphenolic compounds. The purpose of this study is to identify and quantify the phenolic compounds and the FTIR spectrum from buckwheat honey from the Balti region, Republic of Moldova. The radical scavenging activity of honey was 83% in the DPPH• reaction system. Phenolic compounds from honey were identified by HPLC-MS method, the significant concentrations being lignans, catechins, isorhamnetin, rutin, apigenin and luteolin. FTIR analysis revealed both the carbohydrate profile in buckwheat honey and the presence of active compounds.

demonstrated the antibacterial potential of the buckwheat honey in nosocomial infections including Clostridium difficile, due to the high concentration of phenolic compounds and the synergistic contributions of saturated sugars (~ 80%) The rutin, a flavonol with cytoprotective, vasoprotective, anticarcinogenic, neuroprotective and cardioprotective properties [10,13] is present in buckwheat in larger quantities than in other plants. The ability of honey to scavenge the superoxide anion is used both in the food industry and in the pharmaceutical industry.
The purpose of this study is to identify and quantify the main polyphenolic compounds present in buckwheat honey from the Balti, a region of Republic of Moldova, in 2012. This study is the first to perform an analysis of flavonoid compounds present in the buckwheat honey from the Balti region, Moldova.
The infrared spectroscopic analysis (FTIR,) was used to determine the specific spectral fingerprint, thus being able to construct a model capable of predicting the physico-chemical parameters according to the obtained results. Also, by analyzing the FTIR spectrum, one can appreciate the antioxidant capacity of the respective honey [14,15].

2.1.Material
The study was carried out on buckwheat honey purchased from beekeepers from Balti region, the Republic of Moldova, in 2012.

Analysis of total polyphenols content
The total polyphenol content (TPC) was determined by spectrophotometric analysis, using T80 UV/VIS spectrophotometer and gallic acid as standard, according to the method described by the International Organization for Standardization (ISO, 14502-1) [16] . Honey (5 g) was diluted to 50 mL with distilled water. One millilitre of this honey solution was mixed with 5 mL of Folin-Ciocalteu reagent (1:10) and 4.0 mL of sodium carbonate solution (7.5% w / v). After keeping the tubes at room temperature for 60 min, the absorbance at the wavelength λ = 765 nm was read using a UV-Vis spectrophotometer (T80 UV/ VIS spectrophotometer) relative to a control sample (in which 1mL of the extract was replaced). with 1mL of distilled water). The total polyphenol content was expressed as gallic acid equivalents (GAE) in mg/100 g of plant material. The TPC was determined by interpolation of the sample absorbance against a calibration curve built with gallic acid standards (10,20,30,40,50 μg/mL in 10% ethanol) and expressed as milligrams of gallic acid equivalents (GAE) per 100 mg of honey (mg GAE/100 mg), using the calibration curve, y=0.071x+0.006, R 2 =0.0092

Analysis of total flavonoid content
The total flavonoid content (TFC) was determined by spectrophotometric analysis, using T80 UV/VIS spectrophotometer and quercetin as standard, according to the method [17] based on the formation of a yellow flavonoid-aluminum complex and its absorbance reading at 430 nm. To 1mL aqueous extract was added 1 mL 2% AlCl3 atentie (2.5% w / v), 2 mL CH3COOH (10% w / v) and ethanol (70% w / v) to a volume of 10 mL. After 30 min the absorbance at the wavelength λ = 430 nm https://doi.org/10.37358/RC.20.7.8251 is being read. Flavonoid content was expressed as quercetin equivalents, and calculated using the standard curve plotted on the concentration range 10 and 50 μg / mL of quercetin, using the calibration curve, y=0.0045x+0.0062, R 2 =0.00978. All the analysis were performed in duplicate.

Determination of antioxidant activity by DPPH method
The determination of antioxidant activity by the DPPH method was performed according to the protocol of Brand -Williams with some modifications [18]. The stock solution was obtained by dissolving 0.024 g DPPH in 100 mL methanol, was kept in a refrigerator until further use. Prior to analysis, a dilution of 1:10 with methanol was prepared from the stock solution. For analysis, 3 mL of DPPH working solution was added to 0.1 mL extract (1 mg/mL) or the standard solution. The absorbance was measured at 517 nm for a period of 30 min. The absorbance was read at the T80 UV / VIS spectrophotometer, the color of the solution being proportional to the antioxidant concentration.
The percentage of DPPH inhibition was measured according to the following equation: where Acontrol is the absorbance of the control sample. The control contained 100 μL methanol in place of the sample and Asample is the absorbance of the analyzed extracts.

Sample preparation
To a gram of honey, there were added 5 mL of extraction solvent, 1% HCl in methanol. The samples were vortexed for 5 min, sonicated for 30 min, centrifuged for 15 min at 3000 rpm and then filtered. The obtained extracts were stored in the freezer and were used to determine total polyphenols and total flavonoids. The extract prepared for the determination of total polyphenols was filtered through a 0.45 µm filter and injected into the HPLC-DAD-MS Agilent 1200 system.

Analysis of phenolic compounds by HPLC
HPLC-MS analysis of the phenolic compounds by the HPLC-DAD-MS method was performed using the Agilent 1200 HPLC system with DAD detector coupled with Agilent 6110 single quadrupole MS detector. Positive ionization ESI mode was used.
The separation of the phenolic compounds was carried out on a chromatographic column as it follows: Eclipse XDB C18, size 4.6x 150 mm, 5 μm, Agilent, at temperature: 25 0 C.
The mobile phase consisted of: Solvent A: Water + 0.1% Acetic acid / Acetonitrile (99/1) v / v Solvent B: Acetonitrile + 0.1% acetic acid. The gradient used corresponds to the first 2 min mobile phase A 95%, B 5% followed by a linear increase of solvent B to 40% until min 18, linear growth of phase B up to 90% the next 12 min, maintenance for 2 min and return to 95% A and 5% B in the next minute with holding for column balancing 5 min. Total analysis time -30 min with a mobile phase flow rate of 0.5 mL / min Monitored wavelengths: 280nm, 340 nm For MS analysis, the positive ESI ionization mode was used. The ionization parameters were: Capillary voltage: 3000 V; Temperature: 350˚ C, Nitrogen flow rate: 8 l /min. Data acquisition was done in full scan mode in the mass range m / z: 100-1000. The identification of phenolic compounds in samples was done by comparing the retention times, the UV and mass spectra with those of the injected standards.
TFC of buckwheat honey was 47 ± 0.8 mg QE / 100 g sample. TFC of buckwheat honey was about 10 times higher than Polish buckwheat honey (3.23 mg QE/100 g of honey) [23]. Compared to other varieties of honey, buckwheat has a much higher TFC.
The antioxidant activity of buckwheat honey was conducted by DPPH assay, which is one of the most stable free radical. The antioxidant activity of buckwheat honey (expressed as % of inhibition) was 83% . The obtained result can be compared to the study of [24], where the DPPH radical reaction system measured was 87.28% [22]. Compared to the current study, Dzugan  Following the HPLC-MS analysis performed in the present study, the following flavonoid compounds were identified Table 2, Figure 1. Flavonoid quantification, Table 2, was performed based on the calibration curves drawn for each compound, Table 4. The linearity of the method was demonstrated by the correlation coefficient that was for all the compounds analyzed between 0.99-1.  Note: LOD, limit of detection; LOQ, limit of quantification.
The floral origin of nectar and pollen determines the content in phenolic compounds and consequently the bioactive properties of bee honey [30].
It is observed that the highest concentrations were obtained for GCG, EGC, flavonoids that have not been found in other types of buckwheat honey [19,28]. Small amounts of catechins have been identified in heather honey (23.07 μg / g), lavender honey (7.85 μg / g), chaste tree (1.87 μg / g) [29]. This fact shows the possibility of buckwheat honey to be used in different nutraceuticals, knowing the role of catechins in inhibiting bacterial growth [31], the effect of hypocholesterolemia [32,33,34] by lowering cholesterol absorption and increasing fecal excretion and decreasing ROS induced cytotoxicity through intracellular scavenging [35].
Of the total phenolic compounds, 80% are flavonoids, the rest being mainly lignans, namely hydroxisecoisolariciresinol, in a surprisingly high concentration, 97,40 μg / g sample and coumestrol, 4,60 μg / g sample. The level of the main phenolic acids (caffeic, p-hydroxybenzoic, ferulic ) present in other studies [20,21,42 ] are in insignificant quantities. Although in a small amount, coumestrol may exert its stimulation effects on the estrogen receptor, inhibiting adipocyte differentiation [45], known that after pass through the gastric passage, the structure of coumestrol does not change [46,47], and may also act at small concentrations.
ATR-FTIR infrared spectroscopy is a rapid method used for the qualitative characterization of honey. The analysis of the spectrometric fingerprint of honey by determining the chemical bonds specific to the characteristic chemical compounds [48].
In Figure 2 and Table 5 are presented to the FTIR absorption bands, together with the corresponding vibration allocation for buckwheat honey. The spectral domains most often used in honey analysis are those within the fingerprint region, 700-1500 cm -1 , which corresponds to the absorption of monosaccharides, such as glucose and fructose and disaccharides, sucrose [49,50] as well as the region 2500-4000 cm -1 , which characterizes the antioxidant capacity [51]. Table 5.Allocation of the corresponding vibrations for the honey of spectrally made 700-4000 cm -1

Position of the band (cm -1 )
Type and origin of vibration 769,6 δ (C-H) from the structure of carbohydrates  The intense signal was observed at 3237.2 cm -1 where a very large absorption band, characteristic of the O-H bonds, due to the presence of phenolic compounds, was highlighted. Tewari et al. (2005) and Matysek (2018) studied the FTIR fingerprints of several types of honey (buckwheat, clover, alfalfa, carrot, wildflower, orange blossom, sunflower) and found that the absorption band in the area of 3200 cm -1 is the largest for buckwheat, which reveals the highest amount of polyphenols. The FTIR spectral profile of buckwheat in this study is similar to that of buckwheat in Kędzierska-Matysek's study [48].

Conclusions
Phenolic compounds and antioxidant activity can be considered some important factors for honey quality and its functional properties.
Given the results obtained from the analysis of polyphenolic compounds for buckwheat honey used in this research, it is possible to notice that the differences in the content of polyphenols and flavonoids, depending on the geographical area and the floral source.
The results of the study contribute to the knowledge of the major composition of phenolic compounds from buckwheat honey from the Balti region, Republic of Moldova and allow comparison with other honey samples from other countries. Thus, the buckwheat honey from our study is highlighted by much higher amounts of flavonoids compared to phenolic acids. There are high concentrations of GCG, EGC, isorhamnetin.
These determinations made on buckwheat honey from the Republic of Moldova offer opportunities for its use both as a main ingredient in many pharmaceutical and cosmetic preparations, as well as a valuable food for sustaining health.