TOTAL PHENOLIC AND FLAVONOID CONTENT, ANTIOXIDANT ACTIVITY OF FICARIA VERNA

Viktoriia Karpiuk Postgraduate Student at the Department of Technology of Biologically Active Substances, Pharmacy and Biotechnology, Lviv Polytechnic National University, Ukraine e-mail: viktoriia.r.liakh@lpnu.ua, orcid.org/0000-0002-7996-5352, Roksolana Konechna Ph.D., Associate Professor, Lviv Polytechnic National University, Ukraine e-mail: roksolana.t.konechna@lpnu.ua, orcid.org/0000-0001-6420-9063


Introduction
Despite significant advances in the modeling and creation of synthetic drugs, the popularity of herbal therapy is increasing and its competence is expanding. Today, the world pharmaceutical industry is making extensive use of herbal raw materials, which are the basis for the creation of medicines. Medicinal plants contain evolutionarily formed complexes of native substances engaged in complex interactions.
A large number of drugs that are manufactured worldwide have natural ingredients of plant origin. Natural drugs are known to have a milder effect than synthetic agents. Undesirable side effects of drugs, including those of synthetic origin, are observed in 10-40% of patients and are one of the obstacles in the development of new drugs. The percentage of side effects significantly increases during self-medication. Thus in more than 60% of cases of self-medication, there was observed irrational and unjustified use of drugs. So it is promising to expand the range of herbal medical products with new effective plant-based preparations, in particular those based on the herbs widely used in ethnomedicine.
Therefore, the search for new species of plants that could be a source of biologically active compounds, such as flavonoids, coumarins, hydroxycoric acids, alkaloids, saponins, amino acids,and so on. One of the most relevant and promising representatives of the Ukrainian flora to be used in modern medicine and pharmacy is a plant from the family of Ranunculaceae -Ficaria verna. This plant is typical for the ethnopharmacology of East Slavic peoples. It has been used to treat bronchitis, tracheitis, hemorrhoids, skin rashes, acne, diathesis, gingivitis, polyarthritis, stomatitis, and wounds. (Liakh and Konechna, 2021) The complex of bioactive compounds of plant Ficaria verna has diuretic, expectorant, anti-inflammatory and blood purifying properties.
The Ficaria verna growth range extends from Europe and North Africa to West Asia. Plant populations were found in Belarus, Croatia, Germany, Lithuania, Spain, Algeria, Libya, Tunisia, Israel, Turkey, and Georgia. It was introduced into North America. (Karpiuk, et al., 2020) In Ukraine, it is widespread throughout the territory. it occurs in wet forests, mostly deciduous, often along watercourses, in thickets of shrubs. Ficaria verna contains biologically active substances in both primary and secondary synthesis. It consists of saponins, γ-lactones: protoanemonin, anemonin, ascorbic acid (190 mg %),carotene (5,2 mg %). Starch (13.5%), sugars (10%) have been found in underground organs. It also contains triterpenoid saponins. (Hrodzinsʹkyy, 1992) Ficaria verna flowers contain flavonoid compounds (kaempferol 3-O-β-d-(6''-α-1-rhamnopyranosyl)-glucopyranoside (nicotiflorin), apigenin 8-C-β-d-glucopyranoside (vitexin), luteolin 8-C-β-d-glucopyranoside (orientin) and apigenin 8- triterpenes and stearins. (Gudej and Tomczyk,1999) Lactone рrotoanemonin, the main component of the plant, is toxic,but after drying the toxic properties are lost because protoanemonin is converted into anemonin. All parts of the plant contain protoanemonin, but the highest content is found in stems and flowers. Ficaria verna leaves contain fewer flavonoids than flowers. The main components in the leaves are derivatives of the C-glycoside apigenin and luteolin. Ranulinculin and its breakdown products are observed in a raw. ( Tomczyk and Gudej,2003;Tomczyk and Gudej,2002) The purpose of our study is to investigate the chemical composition of the ethanol extracts of Ficaria verna., in particular, phenolic compounds and flavonoids, and to study their and antioxidant effects.

Plant material
Harvesting of medicinal plant material (Ficaria verna. herb, leaves, and flowers) was carried out in ecologically clean regions of west Ukraine in spring 2020. Drying and standardization were carried out according to the requirements of the State Pharmacopoeia of Ukraine. (Derzhavna Farmakopeya Ukrayiny. Dopovnennya 2).

Determination of total phenolic content
The determination was performed using a spectrophotometric analysis using a modified Folin-Ciocalteu method. 0,1 ml of Folin reagent, 1,5 ml of distilled water, and 0,3 ml of 20% Na 2 CO 3 solution were added to 0,1 ml of the analyzed solution, diluted in a ratio of 1:10. Kept for 150 min in the dark place Тhe optical density of the resulting solution was measured at 760 nm. The conversion was performed per gallic acid according to a calibration curve that was constructed under similar conditions, replacing the analyte with the gallic acid solution used as standard. A 3-fold measurement was performed for data validity (Skotti et al., 2014;Krvavich et al., 2019).

Determination of total flavonoid
The number of flavonoids was determined by a modified spectrophotometric method by the complexation reaction of flavonoids with AlCl 3 . For this purpose, a 5% solution of NaNO 2 , a 0.1M solution of sodium hydroxide NaOH, and a 10% solution of AlCl3 were prepared. 0.2 ml of the obtained Ficaria verna. herb extract was taken into a test tube and dissolved in 0.8 ml of ethyl alcohol. 0.06 ml of 5% sodium nitrite solution was added and mixed. After that, the tube was kept for 5 min. 0.06 ml of a 10% solution of aluminum chloride was added and kept for 5 min until the reaction was complete. Then 0.4 ml of 0.1 M sodium hydroxide solution and 0.480 ml of ethyl alcohol were added. After that, the tube was kept for 5 min in a dark place.
The measurements were performed at a wavelength of 510 nm. For calibration, a standard curve was constructed using the solution of quercetin as standard, and the content of flavonoids was determined in terms of quercetin. A 3-fold measurement was performed for the accuracy of the data ( Do et al., 2014).

Determination of the antioxidant effect 2.5.1 DРРH radical scavenging effect
The DРРH method of measuring the antioxidant effect of the extract was used with some modifications. Freshly prepared solution of DРРH was about 0.1 mM (0.2 g DРРH in 500 mL of ethanol). 4.5 mL of solution of DРРH and 500 μL of the extract were mixed in a test tube, which was incubated for 30 minutes in the dark at room temperature. A UV-VIS spectrophotometer was used for measuring the decrease in absorbance (at 517 nm). (Konechna, R.et al.,2017) The following formula was used for calculating percentage of inhibition of the radicals: %inhibition = (Acontrol − Asample) /Acontrol × 100% where Acontrol is the absorbance of DPPHsolution without extract and Asample is the absorbance of the sample with the added DPPH solution. A 3-fold measurement was performed for the accuracy of the data (Do et al., 2014).

Total phenolic and flavonoid contents
The total content of phenolic compounds in the investigated extracts was determined, the result is expressed in mg of gallic acid per g of plant material. The total content of flavonoids was determined, the result is expressed in mg of quercetin per g of plant material. The results are presented in Table 1, Table 2, Table 3. It was found that among the extracts from the herb Ficaria verna the maximum content of both phenolic compounds and flavonoids was observed in 70% of water-ethanol extracts.
The content of flavonoids in the tested extracts ranged from 7,41 to 18,37 mg quercetin/g.The highest value was observed for the FH3 extract, the extractant being 70% aqueous-ethanol solution. It was found that among the extracts from leaves of Ficaria verna the maximum content of both phenolic compounds and flavonoids was observed in 70% of water-ethanol extracts.
The content of flavonoids in the tested extracts ranged from 0.675 to 10,37 mg quercetin/g.The highest value was observed for the FL3 extract, the extractant being 70% aqueous-ethanol solution. It was found that among the extracts from flowers of Ficaria verna the maximum content of both phenolic compounds and flavonoids was observed in 70% of water-ethanol extracts.
The content of flavonoids in the tested extracts ranged from 0.386 to 6.32 mg/g. The highest value was observed for the FF3 extract, the extractant being 70% aqueous-ethanol solution.
The maximum content of phenolic compounds and flavonoids was observed in extracts with the herb Ficaria verna, the lowest content in extracts from the flowers of Ficaria verna.

Antioxidant activity
For the evaluation of the antioxidant activity of single compounds has been widely used relatively stable organic radical DРРH as well as the different plant extracts.
A rapid decrease in the optical density at 517 nm was induced by the addition of extracts to the DРРH solution.
The effect of Ficaria verna extracts of different concentrations in comparison with quercetin and vitamin C on the inhibition of DРРH radical is shown in Table 4, Table 5, and Table 6 Our investigation shows that the free radical scavenging ability of FН1, FН2 -extracts was better than quercetin. The free radical scavenging ability of FН1, FН2, and FН3 extracts were better than Vitamin С.
The results prove that FН1, FН2 extracts improve the scavengers of radical DРРH cations more than vitamin C or quercetin.