APPROACHES TO THE DETERMINATION OF ANTIOXIDANT ACTIVITY OF EXTRACTS FROM BEE BREAD AND SAFFLOWER LEAVES AND FLOWERS

The main objective of this study was to develop approaches for the determination of total antioxidant activity of natural products (bee bread and safflower extracts) using DPPH radical scavenging assay. Considering that analytical procedures and results related to this assay and reported by many authors are significally differed between each other and depend on many factors (the nature of tested extracts, the nature of solvents for extraction, a reaction time of DPPH with a sample, DPPH solvents and concentration, ratio between DPPH and an extract, etc.), the methodology of the evaluation of antioxidant capacity of different origin extracts by DPPH radical scavenging assay was developed. Ascorbic acid (AA) was used as standard antioxidant and the correlation between the percentage of DPPH scavenging and AA concentration was determined at two different initial absorbances of DPPH solution. Average concentration of AA which inhibited 50% of DPPH radicals (IC50) was equal to 156.0 – 171.26 μg.mL . The reaction kinetics of DPPH inhibition by bee bread and safflower extracts was described by the curves of the dependence of the total antioxidant activity on time with squared correlation coefficients (R) in the range of 0.89 – 0.98. The reaction times for these extracts were from 40 to 70 min at the correct ratio of volumes between the tested extracts and a DPPH solution. These studies demonstrated that the extracts obtained from bee bread of 2016 year of pollen collection had significantly higher the total antioxidant activity compared with the extracts of bee bread of pollen collection of 2015 considering the ratio of bee bread and the solvent in the extracts and volume of the extract for the procedure. This fact is explained not only botanical origin bun also the time of the storage of bee bread before the preparation of extracts. There was not found significant differences in the total antioxidant activity of extracts from flowers of safflower sown in fall and in spring. Antioxidant activity of the extracts from leaves of spring sown safflower was higher compared with the total antioxidant activity of the extracts from fall sown plants. Ascorbic acid equivalents of the tested extracts could be ranged as follows: bee bread of 2016 pollen collection >bee bread of 2015 pollen collection >leaves of safflower spring sown >flowers of safflower spring sown >flowers of safflower fall sown >leaves of safflower fall sown.


INTRODUCTION
Herbal preparations and food of plant origin are considered to be the most promising sources of antioxidants. In the initial stages of the development of herbal preparations or food herbal products, especially from nonpharmacopeia plant material, the determination of the sum of such analytical markers as phenolic compounds, provitamins (carotenoids) and vitamins (vitamin C, vitamin E (tocopherols)), microelements with changeable valence (manganese, iron, selenium etc.), amino acids and enzymes, having antioxidant properties, unsaturated acids could generally define biological value including nutritional properties of these products. Se . The high phenolic and flavonoid content is responsible for the bioactivity of crude extracts. Flavonoids are highly effective scavengers of most oxidizing molecules, including singlet oxygen, suppress reactive oxygen formation, chelate trace elements involved in free-radical production, scavenge reactive species and up-regulate and protect antioxidant defenses (Baba and Malik, 2015). The presence of a double bond between C2 and C3 in the C-ring in a flavonoid structure influences antioxidative properties. A carbonyl group at the C4 position enables the compounds to scavenge hydroxyl radicals. This group is presented in flavons, flavonols, flavanons, and isoflavоns. The ability to scavenge hydroxyl radical increases with the number of hydroxyl groups present in the B-ring, especially at the positions C3′ and C4′. The presence of hydroxyl groups at the C5 and C7 positions in the A-ring, C3′ and C4′ in the B-ring, as well as C3 in the C-ring enhances the inhibition of lipid peroxidation Polyphenols are components of safflower and bee pollen that determine their antioxidative activity as they scavenge free radicals and chelate metal ions. Flavonoids are the largest group of phenolic compounds, which have antioxidant, antimicrobial, antitumor, antiinflamatory activities, regulate free-radical reactions in the body. The pollen flavonoids quercetin, rutin and chrysin demonstrated a chemopreventive activity by increasing apoptosis. Flavonoids in plants constitute the antioxidant system, in which various antioxidants restore each other and exhibit a synergistic effect ( Bee bread is a fermented mixture of plant pollen, honey, and bee saliva that worker bees use as food for the larvae, and for young bees to produce royal jelly. Pollen collected by bees is mixed with a small amount of honey and saliva and packed into the cells of the honeycomb where it undergoes a chemical change (lactic fermentation) . Different modifications and optimizations of DPPH assay are performed with the purpose of their adaptation to tested samples or are the invention of researchers. Therefore, it is not easy to compare results of different scientists.
Total antioxidant activity of extracts of bee bread and leaves and flowers of safflower may serve an important marker on the initial stages of the DPPH assay development, including stage of their extracts obtaining (choice of an optimal solvent for extraction, duration of extraction, the type of extraction, the time of collection of bee bread etc.). Therefore, the main objective of this study to develop approaches for determination of the total antioxidant activity of extracts from bee bread as well as leaves and flowers of safflower sown in fall and spring by means of DPPH and evaluate the differences in antioxidant activities of the tested extracts.

MATERIAL AND METHODOLOGY Biological material
Bee bread was obtained from Mykolajev and Poltava regions of Ukraine. Samples were collected in the summer (July) of 2015 and May -June of 2016. Three samples of bee bread were collected in Mykolajev region and one sample in Poltava region. Leaves and flowers of safflower were collected in July 2016 in Chisinau area of the Republic of Moldova. Leaves and flowers were collected from at least 10 safflower plants of fall as well as spring sowing. The extracts were prepared from medium sample of identical plant material in 5 replicates.

Ethanol extraction
Some bee bread samples in the amount 2.5 g were extracted with 50 mL of 50% ethanol for 9 days, or in the amount 10.0 g were extracted with 100 mL of 50% ethanol for 21 days. The process of the preparation of bee bead extracts is described in Table 1. Supernatants were decanted and filtered through filter paper and stored in fridge (2 -8 °C). 1, 7-11 batches of extracts were prepared from bee bread samples collected in Mykolajev region and 3 batch was prepared from bee bread collected in Poltava region. 7, 8, 9 and 10, 11, respectively, were prepared from different granules of the same example.
The extraction from leaves and flower of safflower was performed by maceration procedure using 70% aqueous ethanol. Ratio between the fresh plant material and ethanol solution was 1 : 10 for leaves and 1: 20 for flowers. After 7 days of extraction, the extracts were filtered through paper filter and stored at 4 -6 °C.

Potentiometric determination of pH
Values of pH of bee bread samples was determined by pH meter WTW 3110 (Germany) previously calibrated with buffers pH = 4.01 and pH 6.86. 2% suspensions of three samples of bee bread were used for determination of their value of pH.

DPPH free radical scavenging assay
Total antioxidant activity of samples was measured using 2,2-diphenyl-1-picrylhydrazyl (С 18 H 12 N 5 O 6 ) (DPPH) according to modified analytical procedure (Baba and Malik, 2015).The extracts or their appropriate dilutions of bee bread (0.05 mL) were mixed with 1.95 mL of DPPH solution (0.0025 g DPPH in 100 mL 50% methanol) and incubated in the darkness for 60 -70 min. Every 10 min the absorbance of the reaction mixtures was determined using the UV/Vis spectrophotometer (Genesys 20, USA) at 515 nm. The blank of a sample consisted of 0.05 mL of the corresponding extract or its appropriate dilution and 1.95 ml of 50% methanol. The blank of DPPH solution consisted of 0.05 mL of 50% ethanol and 1.95 mL of 50% methanol.
This analytical procedure was tested on leaves and flowers of safflower in the same way with one exception: initial extracts (0.05 mL) were mixed with 1.95 mL of DPPH solution. For positive control 0.05 ml solutions with diferent concentration of ascorbic acid were mixed with 1.95 mL of DPPH solution (at two different absorbances of DPPH solution). No. 1/2017 All the tests were performed in triplicate. The radical scavenging activity of a tested extract or its dilution was calculated as follows: where: A (DPPH)the absorbance of a solution DPPH; A (extract)the absorbance of a sample or its dilution. Every 10 min absorbances of the solution DPPH and the tested sample were measured.
Ascorbic acid (AA) was used as standard antioxidant, its calibration curves were determined in the range of concentrations from 0.04 to 0.4 mg.mL -1 at the different initial absorbances (Figure 1). The squared coefficients of correlation of the linear equation for these curves were 0.9371 and 0.9755 that permitted to express the obtained results of the total antioxidant activity of the tested extracts in equivalents of ascorbic acid. Concentration of AA which inhibited 50% of DPPH radicals (IC 50 ) was equal to 163.5 ±7.5 µg.mL -1 . .5 -5.0. It seems that the limits of Ukrainian standard are too wide. It is suppose that bee bread with pH higher than 4.3 can be of poor quality because of low content of lactic acid.

Values
One of the methods for assessing the total antioxidant activity is determination of the absorption of the solution of DPPH, dissolved in methanol or etanol of certain concentration in water, without addition of a sample (a herbal product) and with the addition of this sample at specific intervals of time.
The reduction of a DPPH absorbance with compounds with antioxidant properties (compounds containing hydrogen) changes purple color of a reaction mixture into slightly purple or even pale or yellowish depending on the content of antioxidants in a sample and their nature. Fifty percent ethanol was chosen as a solvent for the extraction from point of view of the extraction of both hydrophilic and hydrophobic compounds from bee bread simultaneously. In addition to this, some literature data confirm that aqueous and 50% ethanolic extracts have higher antiradical scavenging activity compared to 90% ethanol (Eswaran and Bhargava, 2014).The studies have been started with the selection of the methanol concentration for dissolution of DPPH. The studies conducted on two native extracts of bee bread demonstrated the dependence of radical scavenging activity on methanol concentration in solvent for DPPH.
As below provided results show (Figure 2), the total antioxidant activity of bee bread extract of batch 1 in the presence of 100% methanol is lower. In addition to it, the obvious visible precipitation was observed for batch 1 using 100% methanol as a solvent of DPPH. It is supposed that 100% methanol as a solvent of DPPH precipitates more hydrophilic substances with antioxidant activity. This assumption is agreed with literature dates that one of the major drawbacks of measuring antioxidant activity by means of DPPH is missing a large contribution of antioxidant activity from proteins and other hydrophilic antioxidants because they are precipitated by methanol (Nicklisch and Waite, 2014). Among more hydrophilic antioxidants could be flavonoids in the form of glycosides with 3 monosaccharide remnants or proteins. These flavonoids are soluble in water but not soluble in strong alcohols (Anonym, 2009).
The next stage of the elaboration of the analytical procedure of total antioxidant activity measuring by means of DPPH was to select the volume of a sample for the determination of the reliable total antioxidant activity of extracts. Figure 3 demonstrates that if the concentration of antioxidants is too high, then DPPH is reduced very fast. In that case, the kinetic curves of antioxidant activity changes on time are more parallel to axis X (time) and what is more the total antioxidant activity is equal 80 -85% and does not depend on the volume of the extract (20 -50 mkl). These studies were conducted for batch 7. The same situation was observed for positive control: at too high ascorbic acid concentration (0.4 -0.6 mg.mL -1 ) the part of kinetic curve of antioxidant activity change on AA concentation is parallel to axis of concentration.
Another stage of our study was to check antioxidant activity of bee bread extracts using 0.0025% DPPH Figure 1 Dependence of the total antioxidant activity on concentration of ascorbic acid: 1curve at the initial absorbance of DPPH 0.603 (R² = 0.9371); 2curve at the initial absorbance of DPPH 0.520 (R² = 0.9755).

Figure 2
The dependence of the antioxidant activity of two extracts of bee bread on the concentration of methanol in DPPH solution.

Figure 3
The dependence of the total antioxidant activity on the volume of the native bee bread extract (the initial absorption of DPPH was approximately 0.500). solution with date of its storage for 24 hours. These studies showed that absorption of a DPPH solution was reduced with the time of its storage and the total antioxidant activity of extracts dilutions increased, respectively (Figure 4). In these studies the following regularity was observed: the less the absorption of a DPPH solution the more the total antioxidant activity of a sample was. Figure 4 shows that antioxidant activity of the extract dilution of batch 8 is significantly higher at the less initial absorption of DPPH 0.471 (80.51%) compared with 64.25 -68.64% at the initial absorption of DPPH 0.587 -0.592. There is the same situation with batch 9. On the base of obtained results, it is concluded that it is possible to compare results of measuring the total antioxidant activity only at the same values of the initial absorption of a DPPH solution. Consequently, the total antioxidant activity of batch 8 is higher than that of batch 7 if we take into consideration the initial absorpion of DPPH 0.466 and 0.471, respectively. Similarly, the total antioxidant activity of batch 9 is higher than that of batch 8 if we take into consideration the initial absorpion of DPPH 0.576 and 0.587 or 0.592, respectively.
One more stage of the development of analytical procedure of measuring the total antioxidant activity was approbation of the developed procedure for assay of the total antioxidant activity of safflower leaves and flowers extracts. The results are presented in Figure 5.
The reaction kinetics of DPPH inhibition by safflower extracts is good described by the curves of the dependence on time with squared correlation coefficient in the range of R 2 = 0.89 -0.98. The reaction times for these extracts were in the range from 40 to 60 min. As figure 4 indicates that total antioxidant activity of the extract from leaves of spring sown safflower is much higher compared with flowers spring sown safflower. It is explained by the ratio between the fresh plant material and ethanol: 1: 10 for leaves and 1: 20 for flowers. Content of AA equivalents in the extract from leaves of spring sown safflower was higher, but no significantly, than that in the extract from leaves of fall sown plants. Content of AA equivalents in the extract from flowers of spring sown safflower was similar with that in the extract from flowers of fall sown plants. Considering that the antioxidant activity is predetermined by accumulation of secondary metabolites on plants, the obtained data correlated with the content of total polyphenols and flavonoid glycosides, in safflower plants of spring and fall sown (Ivanova et al., 2016). The total phenolic content in flower extracts collected from plant sown in fall and in spring was similar (0.25 ±0.02 mg.mL -1 ). Leaves of spring sown safflower contained the total polyphenols as well as the flavonoid glycosides more by 20 -30% than leaves of fall sown plants.
Despite fact that the ratio between fresh plant materials and ethanol is 1: 10 for leaves compared to flowers 1:20, the total antioxidant activity of the extract from leaves of spring sown safflower is only higher 1.67 times compared with flowers spring sown safflower.The same situation is observed with the extracts from leaves and flowers of fall sown safflower (71.46 : 42.41 = 1.68) ( Table 2). Ascorbic acid equivalent of antioxidant activities of tested extracts could be ranged following: leaves of safflower spring sown ≥flowers of safflower spring sown ≥flowers of safflower fall sown >leaves of safflower fall sown. The present study revealed that the extract from leaves of safflower sown in spring possessed the greatest antioxidant activity in ascorbic acid equivalent.
In these studies we also observed the following regularity: the less the absorption of negative control was the higher the total antioxidant activity of extract from leaves or flowers of fall sown safflower was (Table 2). For comparison, the total antioxidant activity of extract from flowers of fall sown safflower was 34.54% at the initial absorbance of DPPH 0.587 and, respectively, 42.41% at the initial absorbance of DPPH 0.466.
Table 2 also shows that samples of bee bread extracts of samples 7, 8, 9, 10 and 11 had significantly higher total antioxidant activity compared with batches 1 and 3 considering the ratio of bee bread and the solvent in the extracts and volume of the extract for the procedure. This fact can be explained not only botanical origin bun also the time of the storage of bee bread before the preparation of extracts. Considering the same ratio 1:10 of raw material and the solvent in the tested extracts of bee bread and safflower and the same initial absorbance of DPPH (0.589 -0.612), the results of the total antioxidant activity and AA equivalents are comparable for the extracts from leaves of spring sown safflower (67.14%), flowers of The results of AA equivalents in 1 mL of an extract for all the safflower extracts occupy the intermediate place between ones for the two batches of bee bread 1 and 3. Table 2 also demonstrates that the extracts prepared from granules of the same example of bee bread have insignificant distinguished results. The slight difference could be explained by pollen collecion from different flowers in the same time and analysis deviation as well.

Figure 5
The dependence of the total antioxidant activity of the safflower extracts оn the time.

CONCLUSION
In conclusion, the results of this investigation confirm that bee bread and safflower are sources rich in compounds with antioxidant properties. The best results were observed for the samples of bee bread with a shorter time of storage. The further studies will be directed on the establishment of correlation of the total antioxidant activity and total phenolic contents and flavonoids.