PARAMETERS OF ANTIOXIDANT ACTIVITY OF GALEGA OFFICINALIS L. AND GALEGA ORIENTALIS LAM. (FABACEAE LINDL.) PLANT RAW MATERIAL

The plant raw material of Galega officinalis L. (goat’s rue) and Galega orientalis Lam. (fodder galega) investigated in this study. These species are known as fodder crops with high productivity of green mass and as medicine plants. The current study was aimed to evaluate an accumulation in dry raw of selected plants the total content of phenolic acids (TPA) and flavonoids (TFC) as compounds with antioxidant activity (AA) by spectrophotometric method. AA by DPPH-method and phosphomolybdenum method (reducing power (RP)) was measured. Study of ethanolic extracts of G. officinalis showed accumulation of TPA in different organs in range from 3.65 to 15.17 mg.g -1 caffeic acid equivalent (CAE) and TFC from 10.08 to 65.75 mg.g -1 quercetin equivalent (QE), AA by DPPH-method from 6.02 to 8.45 mg.g -1 Trolox equivalent (TE) and RP of extracts by phosphomolybdenum method from 86.56 to 288.15 mg TE.g -1 . In extracts of G. orientalis was identified TPA from 3.52 to 18.52 mg CAE.g -1 and TFC from 6.09 to 46.72 mg QE.g -1 , antioxidant activity by DPPHmethod from 6.80 to 8.48 mg TE.g -1 and antioxidant capacity by phosphomolybdenum method from 52.52 to 188.51 mg TE.g -1 . It was established that less concentration of studied compounds found in the stems for both species. It should be noted that the content of phenolic acids in the leaves was decreased and flavonoids in stems increased during vegetation for both species. Content of phenolic acids in the generative organs and flavonoids in the leaves decreased in raw of G. orientalis during vegetation. Pearson’s correlation analysis demonstrated very strong relations between TFC and AA by DPPH, TPA and RP, TFC and RP for G. officinalis extracts. Very strong correlation in the extracts of G. orientalis found between TFC and RP, TPA and RP. Obtained results can be used in the further biochemical and pharmacological study.


INTRODUCTION
As reported by Meripõld et al. (2017), the first cut of G. orientalis advisable to use as a bioenergy crop and the second cut as forage. Also, fodder galega was the object of allelopathic study. Experimental evidence obtained by Baležentienė (2009)  Nonetheless, it is necessary to carry out a study with plants of a genus of Galega as a source of important biologically active compounds. The aim of this study was to determine the peculiarities of accumulation of compounds with phenolic nature that can detect the antioxidant status of investigated plants as important crops.

Scientific hypothesis
Comparative assessment of the accumulation of phenolic compounds and determination of the antioxidant activity of two species of Galega L. genus during vegetation.

MATERIAL AND METHODOLOGY Conditions of plant growing
The plants were grown in 2017 -2018 at the experimental fields of the M. M. Gryshko National Botanical Garden of the NAS of Ukraine in the Kyiv city (50°24ʹ55ʺN, 30°33ʹ45ʺE).

Biological material
Observation on plants was conducted in the experimental collection of the Cultural Flora Department of M. M. Gryshko National Botanical Garden of the NAS of Ukraine ( Figure 1). Plant raw material of two species -Galega officinalis and G. orientalis were collected in the stages according to Biologische Bundesanstalt, Bundessortenamt and Chemische Industrie (BBCH) coding system (Meier, 2018). According to the BBCH scale, plant samples were taken at the phenological growth stages described for faba bean (Vicia faba L.). Four principal growth stages were assigned: leaf development (19nine or more leaves infolded), inflorescence emergence (50flower buds present, still enclosed by leaves), flowering (65full flowering: flowers open on 5 racemes per plant), and ripening (80beginning of ripening: seed green, filling pod cavity). For chemical analyses plant raw material was dried at 35 °C for three days (Müller and Heindl, 2006). After this, the samples were milled in the powder condition. All biochemical analyses were done in the Slovak University of Agriculture

Sample preparation
For planned analyses, 0.2 g of milling fraction was extracted with 20 mL of 80% ethanol for 24 hours. After centrifugation at 4000 g with Rotofix 32 A (Hettich, Germany) for 20 min, the supernatant was used for measurement (phenolic acids, flavonoids, antioxidant activity by DPPH-method and reducing power of extracts).

Total phenolic acid content (TPAC)
The content of phenolic acids was determined using Farmakopea Polska (1999). 0.5 mL of sample extract was mixed with 0.5 mL of 0.5 M hydrochloric acid, 0.5 mL Arnova reagent, 0.5 mL of 1 M sodium hydroxide (w/v) and 0.5 mL of distilled water. Absorbance at 490 nm was measured using the spectrophotometer Jenway (6405 UV/VIS, England). Caffeic acid 1 -200 mg L -1 (R 2 = 0.999) was used as a standard. The results were expressed in mg.g -1 caffeic acid equivalents (CAE).

Total content of flavonoids (TFC)
Analise was conducted according to the procedure which was described by Shafii et al. (2017). 0.5 mL of sample extract was mixed with 0.1 mL of 10% (w/v) ethanolic solution of aluminium chloride, 0.1 mL of 1 M sodium acetate and 4.3 mL of distilled water. After 30 min. in darkness the absorbance at 415 nm was measured using the spectrophotometer 6405 UV/VIS (Jenway, England). Quercetin 0.01 -0.5 mg L -1 (R 2 = 0.997) was used as the standard and the results were expressed in mg.g -1 quercetin equivalents (QE).

Reducing power of extracts
Reducing power of extracts was determined by the phosphomolybdenum method of Prieto, Pineda and Aguilar (1999) with slight modifications. The mixture of 1 mL of sample, 2.8 mL of monopotassium phosphate (0.1 M), 6 mL of sulfuric acid (1 M), 0.4 mL of ammonium heptamolybdate (0.1 M) and 0.8 mL of distilled water was incubated at 90 °C for 120 min, then rapidly cooled and detected by monitoring absorbance at 700 nm using the spectrophotometer Jenway (6405 UV/VIS, England). Trolox 10 -1000 mg L -1 (R 2 = 0.998) was used as the standard and the results were expressed in mg.g -1 TE.

Statistical analysis
The statistically treated data are given in tables as the arithmetical mean values and their standard errors. Data were submitted ANOVA and differences between means compared through the Tukey-Kramer test (α = 0.05). Correlation analysis was performed using Pearson's criterion.

RESULTS AND DISCUSSION
Antioxidant compounds common nowadays play an important role in protecting factors that explain reducing the risk of different chronic diseases and belong to various classes of biochemical compounds. Phenolic compounds are widespread secondary metabolites in plant extracts, and it possesses various biological activities such as antioxidant, anticarcinogenic, antimicrobial, antiallergic, antimutagenic, anti-inflammatory etc. In Table 1 and Table 2 are shown data of accumulation of total phenolic acids and total flavonoids content in vegetative and generative organs of G. officinalis and G. orientalis during vegetation. Polyphenol compounds can form several hydrogen bonds and even ionic bonds with most proteins. They modulate the activity of many proteins, involving enzymes, ion channels, etc. As a consequence, many polyphenols are pharmacologically active, being among antioxidants, anti-inflammatory, antibacterial, antifungal, and antiviral (Wink, 2013). (2014) reported that G. officinalis leaves contain 3.6 mg GAE.g -1 of polyphenol compounds. According to Tusevski et al. (2014), this parameter for G. officinalis plants was 32.53 mg GAE.g -1 . Pehlivan Karakas, Sahin and Türker (2016a) obtained twenty phenolics compounds from methanolic leaves extracts of G. officinalis. The total phenolic content, in this case, was 36.69 mg.g -1 of dry extract. According to Sałata and Gruszecki (2010), roots and leaves in vegetative stages of plants contained considerably more phenolic acids than the beginning of the flowering period, while in leaf nodes more polyphenolic compounds were marked during flowering than at the vegetative stage. Total phenolic acids in plant raw material of investigated G. officinalis plants were in the range from 3.65 to 15.17 mg CAE.g -1 depending on the phase of growth (Table 1).

Maslennikov, Chupakhina and Skrypnik
Flavonoids belong to derivates of simple phenols, and their synthesis increases at the stress conditions due to microbial infections, injury, deficiency of nutrients, changing of temperature, etc. (Kulbat, 2016). Flavonoids are biologically active compounds that possess the ability to capture radicals and play a significant role in agriculture and pharmaceutical chemistry as anti-hyperglycemic, anticancerous, anti-allergic, anti-viral, immune-stimulating activity (Sulaiman et al., 2013; Marella, 2017).
The level of flavonoid accumulation in raw of G. officinalis during vegetation was in the range from 10.08 to 67.75 mg QE.g -1 . The study of Tusevski et al. (2014) resulted that the concentration of flavonoids in this plant was 8.95 ±0.13 mg CAE.g -1 .
As shown in Table 2 phenolic acid content of G. orientalis plant raw material ranged from 3.52 to 18.52 mg CAE.g -1 during vegetation. The concentration of flavonoids ranged from 6.09 to 46.72 mg QE.g -1 . According to Baležentienė (2009) report, the highest total content of phenols was determined at the budding stage which was characterized as the most intensive growth period of the plant shoot.
Also, it should be noted that minimal content of phenolic acids and flavonoids in the plant raw material of both investigated species during vegetation was identified in stems. Obtained data showed that higher accumulation of investigated biochemical parameters was different for two species. So, flavonoids content for G. officinalis were maximal in inflorescences and phenolic acidsin leaves (inflorescence emergence stage). For G. orientalis total content of phenolic acids and flavonoids was maximal in leaves (inflorescence emergence stage).
There are a great number of methods for the determination of antioxidant capacity based on different principles. One of them is the DPPH method that is rapid, simple, and accurate (Marinova and Batchvarov, 2011; Pisoschi et al., 2016). The method is based on the scavenging of DPPH through the addition of a radical species or antioxidant that decolorizes the radical solution (Saeed, Khan and Shabbir, 2012). Trolox Equivalent Antioxidant Capacity assay is widely used to evaluate the antioxidant property of investigated products (Kumar, Sharma and Vasudeva, 2017). Figure 2 demonstrates the antioxidant capacity by DPPH-method of plant raw material of Galega species during vegetation. We found that the antioxidant activity of investigated ethanol extracts of G. officinalis was in the range from 6.02 to 8.45 mg TE.g -1 . For G. orientalis extracts this parameter ranged from 6.80 to 8.48 mg TE.g -1 .
Also, the antioxidant capacity can be illustrated by the reducing power of investigated extracts as an important indicator. According to some studies, there is some connection between antioxidant activity and reducing power ( Correlation analysis was used to explore the relationships between the polyphenols, phenolic, flavonoids compounds and antioxidant capacities (by DPPH and phosphomolybdenum methods) measured for all plant extracts of Galega officinalis and Galega orientalis ( Table  3, Table 4). The results of this study have demonstrated that investigated antioxidant components in two species of Galega L. had a correlation between different parameters of an experiment during vegetation. In the period of inflorescence emergence we found a very strong correlation between TPA and TF for both G. officinalis and G. orientalis (0.985 and 0.950 respectively). Very strong correlation found between AA by DPPH and RP of extracts for G. officinalis. The relation between TPA accumulation and RP and TFC and RP was moderate for G. officinalis (0.433 and 0.583 respectively). For G. orientalis in this period between TPA and RP was strong relation (0.750) and between TFC and RP very strong (0.919). In the period of flowering indicated that very strong correlation found for both species between TFC and RP. Dramatically strong correlation detected between TFC and AA by DPPH (0.999) for G. officinalis in this stage. However, in this case for G. orientalis found a very weak correlation (0.033). Also, very strong relations found between TPA, TFC and RP for G. officinalis (0.924 and 0.851 respectively). In this case values of coefficient of correlation were 0.670 (strong) and 0.953 (very strong) for G. orientalis. Different values found for two species regarding the relation between AA by DPPH and RP (very strong for G. officinalis and weak for G. orientalis). .55 e Note: Means in columns followed by different letters are different at p = 0.05; each value represents the mean of three independent experiments (±SD); GAEgallic acid equivalents; CAEcaffeic acid equivalents; QEquercetin equivalents.

Figure 4
The reducing power of ethanol extracts of Galega officinalis L. and G. orientalis Lam. during vegetation. Note: LDleaf development; IE-linflorescence emergence, leaves; IE-sinflorescence emergence, stems; IE-binflorescence emergence, buds; F-lflowering stage, leaves; F-sflowering stage, stems; F-flflowering stage, inflorescences; R-lripening, leaves; R-sripening, stems; R-frripening, fruits; means in columns followed by different letters are different at p = 0.05; each value represents the mean of three independent. experiments (±SD). At the period of ripening determined the very strong correlation between TPA and TFC, TFC and RP for both investigated species. Correlation between TPA and RP was moderate for G. officinalis (0.551) and very strong for G. orientalis (0.880). It should be noted that for both species was found a negative correlation between TPA and AA by DPPH, TFC and AA by DPPH, and between AA by DPPH and RP. The direction of correlation between antioxidant components depends on them nature, thus, different types of phenolics possess different antioxidant activity (Vamanu et al., 2011).
According to previous studies, it should be noted that phenolic extracts exhibited different antioxidant activity that depends on their structure (Tatiya et al., 2011). The study of relationships between phenolic compounds and antioxidant activity demonstrated a significant correlation

CONCLUSION
Based on the results obtained in this study concluded that two investigated species of Galega L. as medicine and forage cultures characterized by plant raw material with high antioxidant activity. The maximal content of phenolic acids for both investigated species was found in the leaves in the period at the inflorescence emergence, flavonoids in inflorescences at the flowering stage for G. officinalis and in the leaves at the inflorescence emergence period for G. orientalis. The least content of phenolic acids and flavonoids identified in the stems of both investigated species. Ethanolic extracts of stems of G. officinalis and G. orientalis plants exhibited the most antioxidant activity at the ripening stage by DPPH-method. Reducing power of ethanol extracts was higher for G. officinalis in the buds, for G. orientalis in the inflorescences. Pearson's correlation analysis (at p <0.05) showed very strong values of coefficient of variation between TFC and AA by DPPH (0.999), TPA and RP (0.924), TFC and RP (0.828) for G. officinalis depending on the stage of growth. The highest correlation found in extracts of G. orientalis between TFC and RP at every investigated stage (0.919, 0.953, and 0.959), between TPA and RP (0.880). Obtained results demonstrated that two investigated species of Galega are a good source of antioxidant compounds with polyphenol nature such as phenolic acids and flavonoids. These data can provide further information about an accumulation of flavonoids and phenolic acids in Galega spp. raw that possess antioxidant activity and also can be used in pharmacological investigations.