1. INTRODUCTION:

The use of medicinal plants in the treatment of various diseases has a thousand-year history and does not lose its relevance today¹. According to WHO expert estimates, in many countries the demand for plant raw materials for the production of medicines is constantly growing.

Despite great success in creating synthetic medicines, the popularity of herbal medicine continues to grow. In recent years, Ukraine has seen an increase in the range of medicinal herbal remedies. Ukrainian producers are actively developing new herbal remedies and can offer them to consumers at a lower price than imported ones^{2, 3}.

To introduce into practice new promising types of medicinal plants that can be used for therapeutic and preventive purposes, scientists often study the experience of traditional medicine⁴^,5.

Promising plants for research are representatives of the buttercup family (Ranunculaceae L.), which is distributed all over the world and has about 50-60 genera of plants. The most famous genera to which a significant part of medicinal plants belong are Aconitum, Actaea, Adonis, Delphinium, Helleborus, Nigella, Ranunculus, etc.⁶^,7.

The object of this research is a representative of the genus Buttercup (Ranunculus L.)- Marsh kaluzhnitsa (Caltha palustris). It is distributed in the Ukrainian Carpathians in all high-altitude areas. It grows on the banks of water bodies, wet meadows, lakes, swampy forests, along swamps, along rivers in slow-flowing and stagnant waters, near wet channels⁸^,9.

The plant has been used in traditional medicine in different countries, as it has a wide range of medicinal properties and has anti-inflammatory, bactericidal, antimicrobial, analgesic, diuretic properties. Caltha palustris contains tannins, glycosides (γ –lactones protoanemonin and anemonin), saponins, berberine, bitterness, vitamin C, triterpenoids (palustrolide, caltolide, hederagenic acids), steroids (sitosterol), carotenoids (3-epilutein), coumarins (scopoletin, umbelliferon), choline, carotene, flavonoids, and alkaloids¹⁰.

The plant is not included in the state pharmacopoeia of Ukraine, as well as in the pharmacopoeia of other European countries. Pharmacognostic and pharmacological research of this plant is appropriate, since today a limited number of scientists have been engaged in the study of this plant, in particular identification, quantitative determination and research of pharmacological properties in Ukraine¹¹. At the same time, there are practically no published results of studies of domestic raw materials Caltha palustris on the content of phenols and flavonoids, the study of antimicrobial action, in particular in relation to clinical strains of microorganisms, as well as antioxidant action¹².

The works of some foreign scientists present the results of studying the content of biologically active substances and the use of Caltha palustris in the composition of medical and cosmetic products^13,14.

The aim of the study is to determine the content of phenolic compounds and flavonoids in extracts of the herb Caltha palustris and to study their antioxidant, antimicrobial effects and to study lipid peroxide oxidation and oxidative modification of proteins in rat liver homogenate under the action of ethanol extracts of Caltha palustris.

2. RESEARCH PLANNING (METHODOLOGY):

To achieve this goal, the following stages of research have been identified

1. Preparation of water-ethanol (20%, 40%, 70% and 90%) extracts of Galiha palustris

2. Determination of the quantitative content of phenolic compounds and flavonoids

3. Condocting of the research on the antioxidant and antimicrobial effects of Caltha palustris extracts.

4. Study of lipid peroxide oxidation and oxidative modification of proteins in rat liver homogenate

5. Analysis of the obtained results and identification of opportunities for their further use.

Figure 1. Stages of research of plant raw materials Caltha palustris

3. MATERIALS AND METHODS:

Plant raw materials of Caltha palustris were harvested in places of natural growth (an ecologically clean region of the Volyn region, Ukraine) in 2020 during the growing season, taking into account the specifics of its harvesting. Caltha palustris was dried by air-shadow method, its quality was determined according to the basic requirements of HFC and crushed to a particle size of 3 mm.

Extracts from Caltha palustris were obtained by maceration. A water-ethanol mixture with an ethanol concentration of 20 % (CP1 extract), 40 % (CP2 extract), 70 % (CP3 extract) and 90 % (CP4 extract) was used as an extractant. The ratio of raw materials to extractant was 1:10¹⁵.

The phenolic content was determined by spectrophotometric analysis using the modified Folin-Chokalteu method. 0.1 ml of Folin reagent, 1.5 ml of distilled water and 0.3 ml of 20% Na₂CO₃ solution were added to 0.1 ml of the test extract solution (diluted with the corresponding extractant solution in a ratio of 1:10). The resulting mixture was kept for 150 minutes in a dark place ¹⁶.

The optical density of the resulting solution was measured at 760 Nm on a Specord M 40 spectrophotometer. The conversion was carried out according to the calibration curve for gallic acid, which served as the standard. The study was conducted three times¹⁷.

The quantitative content of flavonoids in the studied extracts was determined using a modified spectrophotometric method using the reaction of complexation of flavonoids with aluminum chloride. For this purpose, the next solutions were prepared: 5% sodium nitrite solution, 0.1 m sodium hydroxide NaOH solution, and 10% AlCl₃ solution. 0.8 ml of ethyl alcohol and 0.06 ml of 5% sodium nitrite solution were added to 0.2 ml of the test extract solution and mixed. After that, the test tube was kept for 5 minutes at ambient temperature and 0.06 ml of 10% aluminum chloride solution was added and kept for 5 minutes until the reaction was completed. After that, 0.4 ml of 0.1 m sodium hydroxide solution and 0.480 ml of ethyl alcohol were added. The resulting solution was kept for 5 minutes in a dark place ¹⁸.

Measurements were performed at a wavelength of 510 Nm on a Specord M 40 spectrophotometer. The flavonoid content was determined in terms of quercetin. For calibration purposes, a standard curve is constructed using quercetin solution as the standard. The study was conducted three times¹^{9, 20}.

The study of the antioxidant effect was carried out using a modified method. At the first stage of the study, a freshly prepared solution of 0.1 mm DPPH was obtained. In the next step, 4.5 ml of DPPH solution was added to 0.5 ml of the test extract and incubated for 30 minutes in a dark place at room temperature. After that, studies were conducted on a Hitachi U-2810 spectrophotometer at a wavelength of 517 Nm. Ethanol was used as a control sample.

The calculation of antioxidant activity was carried out by the formula:

(A control – A sample)

% Inhibition = -------------------------- x 100 %

A control

(1)

Where: A control- optical density of the initial DPPH solution, A sample- optical density of the sample with the DPPH solution. A 3-fold measurement was performed ^{21, 22}.

The effect of free radical uptake of the extract was determined by ABTS cation radical discoloration assay. The base solution was prepared by adding 10 ml of 0.014 mM ABTS solution to a potassium persulfate solution (prepared by dissolving 0.0135 g K₂S₂O₈ in 10 ml of water). The resulting mixture was mixed and left in a dark place at room temperature for 20 hours. Thus, the ABTS cation radical (ABTS*) was obtained. After that, we prepare a diluted solution of ABTS* (A control) - 1 ml of the solution is diluted to 100 ml with water. The test extract was mixed with a dilute ABTS* solution (A sample ) and the absorption coefficient was measured ²³.

The study was performed on a Hitachi U-2810 spectrophotometer at a wavelength of 734 Nm. All measurements were made three times. The percentage of absorption inhibition was calculated using the equation given above.

The radical absorption effect was calculated as % for the test extract compared to the control with ascorbic acid and quercetin²⁴.

The results obtained were statistically processed using STATISTICA 8 and the statistical functions package of Microsoft Excel. The arithmetic mean deviation m, the arithmetic mean value m, Student's t-criteria, the number of repetitions n are determined.

To determine the antimicrobial effect of the studied extract of the herb Caltha palustris, two methods were used: the method of diffusion into Agar (the "Wells" method) using standard nutrient media (MPA, Saburo medium) and the method of serial dilutions (splitting resazurine in broth) ^{25, 26}.

Studies were conducted on standard strains of Staphylococcus aureus (ATCC 25923 (F-49), Escherichia coli (ATCC 25922), Raoultella terrigena (ATCC 33257), Candida albicans (ATCC 885-653), as well as on clinical isolates of microorganisms: Escherichia coli 168 MDR (multidrug resistance), Staphylococcus aureus (MRSA) 23, Pseudomonas putida 182 PDR (Pandrug-resistant), Bacillus cereus 34 (non-MDR), Enterococcus faecalis 26 (MDR and resistant to some antiseptics), Candida albicans 169 (strain resistant to azoles). Clinical isolates of all strains were polyresistant to antibiotics (MDR), according to the European recommendation criteria for assessing the degree of antibiotic resistance, from the Museum of cultures of pathogens of infectious diseases of the Department of microbiology of the Danylo Halytskyi Lviv National Medical University.

The method of diffusion in Agar consisted in introducing 50 µL of the test object into a well with a diameter of 5.5 ± 0.5 mm, with a suspension of the microorganism culture previously applied to the agar plate (McFarland 2.0). The method of serial dilutions (Resazurin Reduction-Based Assay) consists of 50 µL of nutrient medium (Muller-Hinton broth), 100 µL of the test extract and 50 ml of a suspension of the microorganism (McFarland 2.0), with the addition of 15 µL of 0.05% resazurine in each well. All microbiological studies were performed in three repetitions²⁷^,²⁸.

The activity of the extracts was evaluated taking into account the bactericidal effect of ethyl alcohol of the appropriate concentration: K1 -20% ethyl alcohol, K2 - 40% ethyl alcohol, K3-70% ethyl alcohol, K4-90% ethyl alcohol. Testing was performed in three repetitions. Statistical processing was performed using a computer program of the statistical functions package of the Microsoft Excel, 2019 program. The arithmetic mean M, the error of the arithmetic mean m, and Student's t-criteria were determined.

Lipid peroxidation and protein oxidative modification in rat liver homogenate were studied for the extracts that showed the highest activity.

5 ml of potassium-phosphate buffer was added to 0.5 g of thawed and crushed rat liver tissue. 0.3 ml of the test extracts were added to 0.3 ml of the resulting homogenate, and the corresponding solvents were added to the control. To induce lipid peroxidation, 0.3 ml of 2.8% FeSO₄ solution was added and after 10 minutes. 0.3 ml of 4% H₂O₂ solution and incubated for 2 hours. The reaction was stopped with 1.2 ml of 40% trichloroacetic acid, which simultaneously precipitates proteins, and then centrifuged for 10 minutes. at 5000g. Both indicators of oxidative stress were determined in one sample – the content of thiobarbituric acid-active products was determined in the supernatant, and carbonyl groups – in the sediment according to the method of V. I. Lushchak²⁹.

Determination of thiobarbituric acid active products.:

In the selected samples, the content of thiobarbituric acid active products was determined by the reaction of malondialdehyde (MDA) with thiobarbituric acid . At high temperature in an acidic environment, MDA reacts with thiobarbituric acid to form a stained trimethine complex with a maximum absorption of B=532 Nm. 1.5 ml of 0.8% thiobarbituric acid solution in 0.1 m HCl (ph=2.5) was added to 2 ml of the supernatant and incubated in a water bath at 95-100 ℃ for 60 Minutes. After cooling, 3 mL of butanol was added and centrifuged for 10 minutes at 5000g. Extinction measurements were performed in the upper butanol layer at W=532 Nm. The amount of protein in the samples was determined by The Lowry method (Lowry, 1951). The calculation was carried out according to the formula:

E .V₁.V₂

Thiobarbituric acid = ----------- micromole/ mg protein

e .V.C …….(2)

Where: E is the extinction of the test sample; e is the millimolar extinction coefficient (e = 156 cm²/mmol); V1 is the volume of butanol, ml; V2 is the volume of the sample, ml; V is the volume of the supernatant, ml; C is the protein concentration in the supernatant, in mmol.

Determination of the content of carbonyl groups of proteins. The degree of oxidative modification of proteins was determined by the number of additional carbonyl groups formed in the side chains of amino acids, the content of which was determined in reaction with 2,4-dinitrophenylhydrazine. To determine the carbonyl groups content of proteins, 1 ml of a 1% 2,4-dinitrophenylhydrazine solution per 2 M HCl was added to the obtained precipitates after centrifugation of homogenates. The mixture was ground and incubated for 1 hour at room temperature, then centrifuged for 10 minutes at 5000 g. The precipitate was washed three times with 1 ml of a mixture of ethanol and ethyl acetate (1:1) and centrifuged in the previous mode. The washed precipitate was dissolved for 45 minutes in 3 mL of 50% urea solution. The undissolved material was separated by centrifugation in the previous mode. In supernatants, the content of carbonyl groups proteins was determined on an ULAB 108 UV spectrophotometer at a wavelength of I=370 N m (light absorption by 2,4-diphenylhydrazones). The carbonyl groups content was calculated using the formula:

ΔD*V

Carbonyl groups = ---------- nmol/mg protein ……(3)

E₃₇₀

Where: ΔD is the value of the difference between the optical densities of the experimental and control samples (ΔD = Dexp.- Dcontr; V – sample volume (3 ml); E₃₇₀ – molar extinction coefficient of 2,4-dinitrophenylhydrazine (22000m^-1cm^-1); C– total protein concentration, mg/ml.

4. RESULTS:

The first stage of the study was the preparation of extracts from the Caltha palustris. For this purpose, dry raw materials were ground to a particle size of 3 mm and filled with a water-ethanol mixture of the appropriate concentration(20%,40%, 70% and 90%) in the ratio of raw extractant 1:10. extraction was carried out at room temperature by maceration for 1-3 days.

The next stage of the study was to determine the quantitative content of phenols and flavonoids in the obtained extracts. Phenolic compounds are a group of biologically active compounds that have an aromatic ring in the molecule with a hydroxyl or phenolic group and have a wide spectrum of pharmacological activity. Flavonoids are derivatives of phenolic compounds. The amount of phenols in the studied extracts was expressed in mg of gallic acid per g of vegetable raw materials. The amount of flavonoids in the studied extracts was expressed in mg of quercetin per g of vegetable raw materials. These definitions were performed three times. The results of the study are shown in Table 1.

Table 1: Total content of phenolic compounds and flavonoids in Caltha palustris extracts

Extract	Phenols (mg _{Gallic acid}/g) X̅ ± Δ X̅, n=3	Flavonoids (mg _Quercetin/g) X̅ ± Δ X̅, n=3
СР1	19.3±0.11	11.7±0.11
СР2	15.48±0.07	12.6±0.04
СР3	50.51±0.06	19.85±0.08
СР4	27.58±0.17	8.5±0.16

The highest content of phenolic compounds and flavonoids is observed in the extract obtained by maceration of Caltha palustris plant raw materials using an extractant - 70% water-ethanol solution. The lowest quantitative content of phenols was found in 40% water-ethanol extract of the herb Caltha palustris, and flavonoids - in 90% extract of Caltha palustris.

DPPH method and ABTS cationic analysis aimed at determining the free radical absorption properties of Caltha palustris extracts.

Table 2 shows the results of the antioxidant activity of Caltha palustris extract compared to the activity of known antioxidants: quercetin and ascorbic acid.

Table 2: Activity of Caltha palustris extracts in the absorption of DPPH and ABTS radicals.

Extract	Antioxidant activity, DPPH, (%)	Absorption of radical cations, ABTS, (%)
СР1	54.39±0.15	38.59±0.12
СР2	50.87±0.14	36.84±0.09
СР3	77.19±0.16	63.15±0.04
СР4	63.15±0.07	47.36±0.01
Quercetin	59.18±0.07	37.15±0.09
Ascorbic acid	55.36±0.06	31.65±0.15

Analysis of the obtained results of the study indicates a fairly high level of antioxidant effect of extracts of the herb Caltha palustris. It should be noted that the indicators of antioxidant activity of 70% and 90% of water-ethanol extracts of Сaltha palustris are higher than the indicators of antioxidant activity of quercetin and ascorbic acid.

The results of the study of the antimicrobial effect of caltha palustris herb extracts by diffusion into Agar were determined by the presence of Culture growth retardation around the well into which the test extract was introduced. The study data are presented in Table 3.

According to the results of studies, 90% of Caltha palustris extract exhibits selective antimicrobial action against Pseudomonas putida 182 and Bacillus cereus 34. 70% of Caltha palustris extract exhibits selective antimicrobial action against Enterococcus faecalis 26, as well as clinical and reference Candida strains. (Table 3)

The best antimicrobial effect among the tested extracts was shown by water-ethanol (90%) extract of the Caltha palustris (CP4), the minimum inhibitory concentration (MIC) was in a dilution of 1:32 (0.00045 g) relative to Bacillus cereus 34.(Table 4)

Promising for further study are the study of the antimicrobial effect of water-ethanol extracts of Сaltha palustris CP4 against Pseudomonas putida PDR 182 (1:4 (0.0036 g)), which is expressed by the resistance of existing antimicrobial drugs, and CP3 extract (against Candida albicans 139 1:8 (0.0032 g)).

For 70% and 90% of Caltha palustris extracts, which had the highest levels of phenols and flavonoids and showed antimicrobial properties, studies were conducted on lipid peroxidation and protein oxidative modification in rat liver homogenate.

Table 3: Antimicrobial effect of water-ethanol extracts from the herb Caltha palustris

No.	Type	Types of microorganisms	Diameter of the growth retardation zone (мм ± SD)
No.	Type	Types of microorganisms	CP1	К1	CP2	К2	CP3	К3	CP4	К4
1.	Gram-negative bacteria	Escherichia coli (ATCC 25922)	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a
2.		Escherichia coli 168 MDR	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a
3.		Pseudomonas putida 182 PDR	n/a	n/a	n/a	n/a	n/a	n/a	11.1±0.25	n/a
4.		Raoultella terrigena ATCC 33257	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a
5.	Gram-positive bacteria	Staphylococcus aureus (MRSA) 23	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a
6		Staphylococcus aureus (ATCC 25923 (F-49)	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a
7.		Bacillus cereus 34 (non-MDR)	n/a	n/a	n/a	n/a	n/a	n/a	10.1±0.3	n/a
8.		Enterococcus faecalis 26	n/a	n/a	n/a	n/a	14.8±0.3	n/a	n/a	n/a
9.	Fungi	Candida albicans 139	n/a	n/a	n/a	n/a	14.5±0.3	n/a	n/a	n/a
10.	Fungi	Candida albicans (ATCC 885-653)	n/a	n/a	n/a	n/a	9.0±0.4	n/a	n/a	n/a

* n/a: no activity; braking zone diameter (mm), including hole diameter 5.5 mm; data are presented as average ±SD (n=3).

As a result of the conducted studies of lipid peroxidation and oxidative modification of proteins (fig. 1-2) it was found that under the action of 70% and 90% of Caltha palustris extracts, a significant decrease in the content of thiobarbituric acid -active products and the formation of carbonyl protein groups was observed in comparison with the control. This indicates the high antioxidant properties of the obtained extracts.

Table 4: Antimicrobial effect (minimum inhibitory concentration-MIC) of water-ethanol extracts from the herb Caltha palustris

Types of microorganisms	Test extract		Control, alcohol
Types of microorganisms	СР3	СР4	К3	К4
Enterococcus faecalis 26	1:1 (0.0256г)	-	n/a	-
Bacillus cereus 34	-	1:32 (0.00045г)	-	1:4
Pseudomonas putida PDR 182	-	1:4 (0.0036г)	-	1:1
Candida albicans 139	1:8 (0.0032г)	-	1:1	-

`-`: not tested; n/a: no activity

Figure 2. Content of thiobarbituric acid -active products in rat liver homogenate by the action of extracts Caltha palustris (*- р≤0,05; M±m; n=5)

Figure 3. Content of thiobarbituric acid -active products in rat liver homogenate under the action of Caltha palustris extracts

As can be seen from the diagrams, 70% of Caltha palustris extracts were the most effective among the studied 70% and 90% in terms of oxidative stress indicators. When exposed to Caltha palustris extract at a concentration of 70%, there is a decrease in the content of thiobarbituric acid -active products by 68.7 %. Caltha palustris showed slightly less pronounced, although high (compared to the control) antioxidant activity at a concentration of 90%, where we observe a decrease in the content of thiobarbituric acid -active products by 59.4 % (p≤0.05). As for protein oxidative modification, at concentrations of 70% and 90% under the action of Caltha palustris, the content of carbonyl groups of proteins decreased relative to the control by 80.7% and 62.3%, respectively. Consequently, Caltha palustris in concentrations of 70% and 90% significantly reduces the level of lipid peroxidation and protein oxidative modification compared to the control.

5. DISCUSSION:

The obtained research results are valuable for pharmaceutical science, since the object of research Caltha palustris is not a pharmacopoeial raw material and requires a number of studies to develop a pharmacopoeial article. The study also contributes to solving the problem of Pharmaceutical Science in finding new sources of biologically active substances, namely phenolic compounds and flavonoids, which are valuable as antioxidant and antimicrobial agents. This also contributes to the further development of new effective substances based on the complex of biologically active compounds Caltha palustris.

An important aspect of the research is that the herb Caltha palustris is a little-studied object. It is advisable to expand its research on the content of other biologically active compounds and actually deepen the study of the content of phenolic compounds and flavonoids, using other methods and approaches.

Firstly, the results of studies of the antimicrobial effect of extracts were obtained, which are of practical importance, since the study was conducted using not only museum strains, but also clinical ones. Accordingly, this expands the prospects for using extracts of the Caltha palustris in the development of new antimicrobials.

However, it should be noted that it is worth deepening research on the use of various types of plant raw materials of this plant. It should be assumed that there is a potential for a higher quantitative content of phenolic compounds and flavonoids in another type of raw material, for example, leaves or flowers.

6. CONCLUSIONS:

Firstly, the content of phenolic compounds and flavonoids in domestic raw materials Caltha palustris was studied by spectrophotometric method. The highest content of phenolic compounds(50.51±0.01) and flavonoids (19.85±0.01) was observed in the extract obtained by maceration of caltha palustris grass using an extractant - 70% water-ethanol solution.

Antioxidant activity was determined using the DPPH radical (2,2-diphenyl-1-picrylhydrazyl) and the ABTS cation radical (2,2’-Azino-BIS-(3-ethylbenzthiazoline-6-sulfonic acid). It was found that 70% and 90% of water-ethanol extracts of Caltha palustris have the highest level of antioxidant action.

The antimicrobial effect of Caltha palustris extracts was studied. The best antimicrobial effect was shown by 90% extract (for Bacillus cereus 34, Caltha palustris extract 90% (MIC 1:32 (0.00045 g) and Pseudomonas putida PDR 182 (1:4 (0.0036 g)) and Caltha palustris extract 70% ( for Candida albicans 139 1:8 (0.0032 g).

70% and 90% caltha palustris extracts have high antioxidant properties in two indicators of oxidative stress, as they are able to reduce the formation of free radicals in proteins and lipids.

70% and 90% extracts of Caltha palustris on rat liver hepatocytes under conditions of initiation of free radical oxidation in vitro showed antioxidant properties in two indicators of oxidative stress, reducing the level of thiobarbituric acid -active products carbonyl groups of proteins by more than 50% compared to the control.

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Received on 19.12.2023 Revised on 08.06.2024

Accepted on 10.09.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5673-5679.

DOI: 10.52711/0974-360X.2024.00864