Phytochemical analysis and antibacterial activity of different extracts of Filipendula ulmaria (L.) Maxim

Abstract

The aim of this study was to investigate the concentration of phenols, flavonoids and tannins and the antimicrobial activity of methanol, ethyl-acetate and acetone extracts of the species Filipendula ulmaria (L.) Maxim. The antimicrobial activity of the extracts was determined for ten different bacterial species. The total concentration of phenols, flavonoids and tannins in the tested extracts was determined spectrophotometrically. The results show that the amount of tested compounds largely depends on the type of solvent used for extraction from the plant material. The total amount of phenols, flavonoids and tannins was higher in polar solvents compared to the non-polar solvent. The highest concentration of phenols was observed in the flower (74.55 mg GA/g), and the lowest concentration of flavonoids was measured in the root (0.04 mg RU/g). The methanol extract of the flower had the highest, whereas the acetone stem extract exhibited the lowest antioxidant activity. Antimicrobial activity, expressed as a minimum inhibitory concentration, was in the range from 0.312 to 10 mg/ml. The tested Gram-positive bacteria were more sensitive than the Gram-negative bacteria. Based on the obtained results, F. ulmaria was proved to be a rich source of phenols, flavonoids, tannins and antioxidants therefore being a prospective natural antimicrobial agent.

Keywords:
F. ulmaria; phenols; flavonoids; tannins; antimicrobial activity

Introduction

Medicinal plants are being widely used to provide and ensure good health and well-being. Every day people use medicinal and aromatic plants as the oldest form of medicine (Inoue et al., 2019). The species meadowsweet - Filipendula ulmaria (L.) Maxim, the genus Filipendula Mill. (Rosaceae) - has been used by traditional medicine for a long time, but there are still insufficient scientific facts related to this species. There are several possible explanations why this species is known as "meadowsweet" worldwide. The most frequently cited one states that the species was used as a sweetener, as tea or a tincture or as an ingredient to improve aroma (European Medicines Agency, 2011).

In European and Asian traditional medicine, F. ulmaria is used as an analgesic, antipyretic, antirheumatic, astringent, diuretic, tonic, and for the treatment of hemorrhoids and ulcers (Bijttebier at al. 2016., Farzaneh et al., 2022). Filipendula ulmaria has a wide range of applications in traditional medicine, particularly for inflammatory conditions (Samardžić et al., 2018). The anti-inflammatory activity of F. ulmaria can be attributed to the additive or synergistic effects of its bioactive components (such as flavonoid glycosides) and metabolites (Auwera, et al., 2023).

The report of the European Medicines Agency (EMA) on the assessment of F. ulmaria (L.) Maxim. herba and F. ulmaria (L.) Maxim., that the herb (in the form of tea, powder and tincture) and the flowers (as tea) are used for medicinal purposes for oral consumption.

The phytochemical composition of meadowsweet has been the subject of numerous scientific research studies. The fresh aerial part of the plant contains ascorbic acid, phenolic glycosides (up to 6%), essential oils, tannin, mucilage, flavonoids (Braun & Cohen, 2015). Its active ingredients include salicylic acid and its derivatives (salicylaldehyde, methyl salicylate, benzyl salicylate), flavonoids, flavonoid glycosides (quercetin, spiraeoside, hyperoside), tannins (particularly rugosins and tellimagrandins), essential oils, and citric acid (Olennikov et al., 2017; Katanić et al., 2015). Salicylates can constitute up to 70% of the essential oil content of F. ulmaria, depending on the time and conditions of flowering (Ložienė et al., 2023). By using the thin-layer sorbent chromatography method (TLC method) on the extracts of the whole plant, leaves, and flowers of F. ulmaria, rutin, tannic acid, gallic acid, and salicylic acid were identified (Kovaleva et al., 2018).

Scientific research studies have demonstrated that the extract of F. ulmaria significantly reduces capillary permeability (Barros et al., 2013; Shilova et al., 2006), inhibits acetylcholinesterase and tyrosinase enzymes relevant to Alzheimer's and Parkinson's diseases (Neagua et al., 2015), and exhibits pronounced anticoagulant, antiulcer, antidiabetic, and antitumor effects (Barros et al., 2013; Shilova et al., 2006). Boziaris et al. (2011) conducted a research showing that the water-methanol extract of F. ulmaria has antibacterial activity and potential to be used as a natural food preservative against spoilage bacteria and inoculated pathogenic bacteria in fish meat and products.

The results of the study on the nutritional profile of infusions (Olennikov et al., 2016) obtained from the flowers of F. ulmaria, F. camtschatica, F. denudata and F. stepposa indicate that they contain high amounts of phenolic compounds, essential oils, methyl salicylate, salicylaldehyde, and water-soluble polysaccharides belonging to galactans and/or arabinogalactans. Infusions obtained from the flowers of Filipendula species exhibit significant bioactivity, manifested by their ability to inhibit α-glucosidase, as well as notable antioxidant and immunomodulatory properties (Olennikov et al., 2016).

Studies have shown that medicinal plants have been resources of natural antimicrobial compounds, the use of which can be effective in treating infections (Manandhar et al., 2019). Modern medicine is progressively more than willing to accept the use of antimicrobial and other plant-derived drugs. In the last 20 years, the interest in antimicrobial plant-derived drugs has soared, due to the rapid growth rate of bacterial resistance to antibiotics (Cowan, 1999). The extracts of flowers, fruits, leaves, and roots of F. ulmaria species have antibacterial activity against Gram-negative bacteria Pseudomonas aeruginosa but exhibit no activity against Gram-negative bacteria Escherichia coli. The antibacterial effect on Gram-positive bacteria Bacillus subtilis is observed only with extracts of flowers and fruits, whereas extracts of the root, stem, and leaves show no antibacterial activity against this bacterium (Savina et al., 2023).

The aim of this study was: to determine in which parts of the F. ulmaria plant (root, stem, or flower) the highest quantities of phenols, flavonoids, and tannins are found; to identify the most effective solvent (methanol, ethyl acetate, and acetone) for the extraction of these compounds; to ascertain which part of the plant exhibits the highest antioxidant activity in order to identify the parts with potential applications in the pharmaceutical and nutraceutical industries; and to investigate the antimicrobial activity of F. ulmaria leaf extracts against selected bacterial strains, providing insights into the potential use of this plant part in controlling bacterial infections.

Materials and Methods

Botany

Filipendula ulmaria (L.) Maxim is a perennial herbaceous plant. The above-ground stem is erect, angular, and branched in the upper part. The rhizome is sympodial and woody. Leaves are alternately arranged, intermittently pinnately divided, with stipules fused to the leaf blade. Flowers are grouped in terminal inflorescences about 25 cm long. Small flowers are bisexual, polysymmetric, composed of 5 sepals and 5 petals, 20-40 stamens (which are twice the length of the petals), and 6-12 carpels in the gynoecium. The fruit is an achene. The plant blooms from June to August and reproduces by seed (Grlić, 1990).

The natural range of this species encompasses Europe, Western Asia, and Iran. It has been introduced to North America (Colorado, Illinois, Minnesota, New York, Newfoundland, Québec, Vermont and Wisconsin). The plant grows up to 1300 meters above sea level in moderately moist habitats without a dry period. It thrives on soil with a neutral to slightly acidic pH and is moderately rich in mineral content. It is a semi-scioxyphite and belongs to the Euro-Asian floral element.

Plant material and extract preparation

Plant material of F. ulmaria was collected on the mountain Divčibare (19.99213 E; 44.09353 N, 967 m) where geologic base is serpentinized harzburgite. Plant material was identified and the voucher specimen of F. ulmaria was deposited in the Herbarium at the University of Kragujevac, Faculty of Science, Department of Biology and Ecology, with systematic number 35/020. The collected plant material (100 plants) was washed to remove impurities with distilled water. Subsequently, it was separated into above-ground parts, stems, leaves, and flowers. The material was then air-dried in a thin layer at a temperature of around 25°C, protected from direct sunlight, in a well-ventilated room. The dried parts of the plant were ground in a blender and used in the subsequent process. Extracts were prepared using the maceration method. We used 250 ml of solvent and 50 g of plant material. Methanol, ethyl acetate, and acetone were used as solvents. Maceration lasted for three days, after which the obtained extracts were dried using a rotary vacuum evaporator at 40°C. The extracts were stored in a refrigerator at a temperature of +4°C. Table 1 displays the masses of obtained dry extracts and their respective yields.

Table 1.
Masses (g) of dry extracts and yields (%) of roots, stems, leaves, and flowers of the species F. ulmaria

	methanol		acetone		ethyl-acetate
Plant Part	Mass of Extracts (g)	Yield (% w/w)	Mass of Extracts (g)	Yield (% w/w)	Mass of Extracts (g)	Yield (% w/w)
Root	13,46	26,9	11.6	23,2	8.4	16,8
Stem	3.35	6,7	2.63	5,3	1.95	3,9
Leaf	3.1	6,2	2.38	4,7	1.71	3,4
Flower	2.5	5	2.03	4,1	1.59	3,2

Determination of phenolic concentrations in the plant extracts

The concentration of phenols in plant extracts was determined spectrophotometrically as described by Peter et al. (2011). The method is based on measuring the general reducing capacity of phenolic compounds in reaction with the Folin-Ciocalteu phenolic reagent, which consists of a mixture of Na₂MoO₄, Na₂WO₄, HCl, H₃PO₄, and LiSO₄. For the determination of the total phenol content, the following components were used: Folin-Ciocalteu reagent (diluted 10 times with distilled water), a 6% aqueous solution of sodium carbonate (Na₂CO₃), and a methanol solution of the tested extracts. Absorbance readings were performed at λ=725 nm. The total amount of phenolic compounds in the sample was calculated based on the calibration curve equation (the absorbance as a function of concentration) of a standard solution of gallic acid. Three measurements were performed for each sample, and the results were presented as mean values (± standard deviation, SD) in terms of gallic acid equivalents per gram of dry extract (mg GAE/g extract).

Determination of flavonoid concentrations in the plant extracts

The concentration of flavonoids in plant extracts was determined spectrophotometrically as described by Quettier-Deleu et al. (2000). The method is based on the fact that flavonoids form different complexes with metal ions. For the determination of flavonoids, the following components were used: Aluminum (III) chloride (AlCl₃, 2%, in methanol), a standard solution of rutin (1 mg/mL, in methanol), and extract solutions (1 mg/mL, in methanol). Absorbance was measured at λ = 430 nm. A mixture of distilled water and the reagent was used as a control sample. The total amount of flavonoids in the sample was calculated based on the calibration curve of the standard rutin solution. Three absorbance measurements were taken, and the result was expressed as the mean value of three independent measurements (± SD), in terms of rutin equivalents per gram of dry extract (mg RE/g extract).

Determination of tannin concentrations in the plant extracts

The concentration of tannins in the plant extracts was determined spectrophotometrically as described by Porter et al. (1986). In order to determine the tannin content, the following reagents were used: butanol-HCl reagent (95 ml n-butanol and 5 ml concentrated HCl), iron reagent (2 g iron ammonium sulfate dissolved in 100 ml 2N HCl), and extract solutions (1 mg/mL, in methanol). Absorbance readings were performed at λ=550 nm. The quantity of tannins in the sample was calculated based on the calibration curve of the standard solution (y = 0.0095x + 0.006 R^2 = 0.999). Three absorbance measurements were taken for each sample, and the result was expressed as the mean value of three independent measurements (± SD), in terms of cyanidin chloride equivalents (CChE).

Evaluation of antioxidant activity

The free radical scavenging activity in the plant extracts was analyzed using 2,2-diphenyl-1-picrylhydroxyl (DPPH), according to Takao et al. (1994). For antioxidant activity determination, the following reagents were used: a solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) in methanol, a positive control solution in methanol with a concentration of 1000 µg/ml (ascorbic acid and butylated hydroxytoluene were used as positive controls), and extract solutions (1 mg/mL, in methanol). Dilutions were made in order to obtain concentrations of 500, 250, 125, 62.5, 31.25, and 15.62 mg/mL. These solutions were mixed with the prepared DPPH reagent and the samples were incubated for 30 minutes at 25°C in the dark. Triplicate samples were prepared for each analysis and the average value was calculated. The absorbance was measured using a spectrophotometer at 517 nm. Antioxidant activity was expressed as the 50% inhibitory concentration (IC50 values in mg/mL). IC50 values were estimated from the % inhibition versus concentration plot, using a nonlinear regression algorithm. The data were presented as mean ± standard deviation (n = 3).

$$\mathit{\%}\mathit{}inhibition=\left(\frac{Aofcontrol-Aofsample}{Aofcontrol}\right)$$ (1)

Microdilution method

The antimicrobial activity of leaf extracts was investigated using the microdilution method. The antimicrobial activity of F. ulmaria leaf extracts was tested against 10 strains of bacteria including three Gram-positive bacterial strains (Staphylococcus aureus ATCC 25923 and clinically isolated S. aureus, Bacillus cereus) and seven Gram-negative bacterial strains (Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 70063, Pseudomonas aeruginosa ATCC 27853, and clinically isolated E. coli K. pneumoniae, Proteus mirabilis, Salmonella enterica). Bacterial sensibility testing is based on the determination of minimum inhibitory concentrations (MIC). The minimum inhibitory concentration is defined as the lowest concentration of the test compound at which bacterial growth is inhibited. Minimum inhibitory concentrations are determined using the microdilution method as described by Sarker et al. (2007). The microdilution method is performed in microtiter plates with 96 wells under sterile conditions. The density of bacterial suspensions is measured with a densitometer. Suspensions were prepared from the cultures of examined bacteria test strains grown on nutrient agar for 20 h, in sterile saline dilution. For the standardization of bacterial suspensions and determination of bacteria, the McFarland standard number 0.5 was used, which indicates the density of the bacterial suspension (number of bacteria) of 1.5 × 10⁸ CFU/ml. Dilutions of the initial suspension were additionally prepared in order to get a precise value of the bacterial suspension density of 5 × 10⁵ CFU/ml. Then double serial dilutions of the tested extracts were made, with a range from 0.078 mg/ml to 10 mg/ml for the extract and from 0.125 µg/ml to 16 µg/ml for the antibiotic (tetracycline). The total volume in the well was 100 μl. The microtiter plates were incubated at 37 °C for 24 hours. For monitoring the growth of bacteria, resazurin was added. The change of color indicator from purple blue to pink color indicates growth. The lowest concentration, at which there is no change in the color of the indicator, is defined as the Minimum Inhibitory Concentration (MIC). The tested extracts were dissolved in concentrated DMSO (10% of total volume) and then diluted with liquid Mueller-Hinton broth (up to 100% of total volume). The antibiotic was also diluted in a liquid Mueller-Hinton medium. Due to the fact that concentrated DMSO is bactericidal, a solvent control was set confirming that 10% DMSO has no negative effect on bacterial growth.

Data analysis

All measurements (the content of phenols, flavonoids, and tannins in root, stem, leaf, and flower) were carried out in triplicate and expressed as the average value of three independent measurements ± standard deviation. Statistical analyses of data were made by using IBM SPSS Statistics 21.0 (2012). Results were expressed as the mean of three replicates ± standard error (SE). The collected data were analyzed by the one-way ANOVA and Tukey post-hoc test. The difference between groups, regarding the antibacterial activity, was analyzed using a nonparametric Kruskal-Wallis post-hoc test. The statistically significant difference was defined as p < 0.05.

Results

Phenolic content of the extracts

The values of phenolic concentration are presented as gallic acid equivalents, mg GA/g extract (Peter et al., 2011). Results of the total phenolic content in the extracts of the root, stem, leaf, and flower of the species F. ulmaria obtained using methanol, acetone, and ethyl acetate are shown as the average value and standard deviation calculated based on measurements in triplicate (Table 2). The highest concentration of phenols was extracted using a polar solvent (methanol) from the flower (74.55 ± 2.31 mg GA/g), while the lowest concentration of phenols was observed in the extracts obtained with a non-polar solvent (ethyl acetate) from the root (3.61 ± 0.34 mg GA/g). Among all the plant organs studied (root, stem, leaf, and flower), the highest amounts of phenols were detected in the flower. In the methanolic extract of the flowers, the total phenol content (74.55 ± 2.31 mg GA/g) was 30.3% higher than the content extracted with acetone (51.94 ± 2.01 mg GA/g) and significantly greater (81.15%) than the content extracted with ethyl acetate (3.61 ± 0.34 mg GA/g). The total phenol content extracted with methanol in the flower was 64% higher than the one in the leaf, 72.6% higher than the content in the root, and 79% higher than the content in the stem. The obtained values are statistically significant (p > 0.05).

Table 2.
The total amount of phenols determined in different F. ulmaria extracts, presented as equivalents of gallic acid, mg of GA/g extract.

Extract	Flower	Leaf	Stem	Root
Methanol	74.55 ± 2.31a	26.83 ± 0.33a	15.61 ± 0.83a	20.44 ± 2.86a
Acetone	51.94 ± 2.01b	18.83 ± 0.76b	12.77 ± 0.50a	16.66 ± 1.44a
Ethyl-acetate	14.05 ± 0.97c	5.66 ± 0.57c	5.77 ± 0.34b	3.61 ± 0.34b

• Values are mean ± standard deviation of triplicate analyses.
• Values within column followed by the same letter (a, b, c), are not significantly different according to Tukey’s test (p < 0.05).

Flavonoid content of the extracts

Flavonoid concentrations are presented as rutin equivalents (mg RU/g extract) following Quettier-Deleu et al. (2000) and are shown in Table 3. These concentrations are expressed as mean values ± standard deviation. Different letters (a, b, and c) indicate the statistical significance of differences between various plant parts for the same solvent.

Table 3.
The total amount of flavonoids determined in different F. ulmaria extracts, presented as equivalents of rutin, mg of RU/g extract.

Extract	Flower	Leaf	Stem	Root
Methanol	12.53 ± 1.05a	8.18 ± 0.88a	1.16 ± 0.19a	0.43 ± 0.14a
Acetone	9.69 ± 0.76b	5.07 ± 0.45b	2.24 ± 0.04b	0.43 ± 0.07a
Ethyl-acetate	3.49 ± 0.58c	4.57 ± 0.26b	0.25 ± 0.18c	0.04 ± 0.03b

• Values are mean ± standard deviation of triplicate analyses.
• Values within column followed by the same letter (a, b, c), are not significantly different according to Tukey’s test (p < 0.05).

The flower exhibits the highest flavonoid concentration when using methanol (12.53 ± 1.05 mg RU/g) and acetone (9.69 ± 0.76 mg RU/g) as solvents, indicating its richness in flavonoids, particularly with polar solvents. The leaf also contains a significant amount of flavonoids, especially wethanol (8.18 ± 0.88 mg RU/g), although the concentration is lower compared to the flower. In contrast, the stem contains lavonoid concentration lower than the flower and leaf, regardless of the solvent used. The root consistently has the lowest flavonoid concentration, even with a polar solvent like acetone (0.43 ± 0.07 mg RU/g).

These results highlight that the flowers are the richest source of flavonoids in this plant, while the root has the lowest flavonoid content. Moreover, methanol and acetone are more effective for flavonoid extraction compared to ethyl acetate.

Tannin content of the extracts

The values obtained for the concentration of tannins are presented as cyanidin chloride equivalents, mg CChe/g extract (Quettier-Deleu et al., 2000). Results showing the amount of tannins in different parts of the plant (flower, stem, leaf, root) species F. ulmaria, are shown in Table 4, as the average value and standard deviation calculated based on measurements in triplicate.

Table 4.
The total amount of tannins determined in different F. ulmaria extracts, presented as equivalents of cyanidin chloride, mg CChe/g extract.

Extract	Flower	Leaf	Stem	Root
Methanol	38.22 ± 1.41a	13.81 ± 0.27a	8.11 ± 0.72a	9.96 ± 0.89a
Acetone	25.05 ± 0.84b	9.66 ± 0.62b	6.11 ± 0.62b	8.16 ± 0.07a
Ethyl-acetate	7.03 ± 1.01c	4.62 ± 0.39c	2.81 ± 0.16c	1.61 ± 0.07b

• Values are mean ± standard deviation of triplicate analyses.
• Values within column followed by the same letter (a, b, c), are not significantly different according to Tukey’s test (p < 0.05).

The flower exhibits the highest concentration of total tannins when methanol is used as a solvent (38.22 ± 1.41 mg CChe/g), indicating its richness in total tannins. In contrast, the stem contains lower total tannin concentration compared to the flower and leaf, regardless of the solvent used. The root consistently has the lowest concentration of total tannins, even with a polar solvent like acetone (8.16 ± 0.07 mg CChe/g). These results emphasize that the flowers are the richest source of total tannins in this plant, while the root has the lowest total tannin content. Moreover, methanol proved to be the most efficient solvent for extracting total tannins, while ethyl acetate showed the lowest efficiency.

Antioxidant activity of the extracts

Antioxidant activity is expressed as IC50 values (µg/mL) in Table 5, with lower IC50 values indicating greater activity. The flower extract obtained with methanol exhibited the lowest IC50 value (13.27 ± 0.57 µg/mL) compared to other plant parts and different solvents, making it the most potent antioxidant among the examined extracts. The leaf extract also demonstrated high antioxidant activity, particularly with methanol (28.27 ± 0.53 µg/mL) or acetone (64.81 ± 0.01 µg/mL) as solvents. In contrast, both stem and root extracts consistently showed higher IC50 values than the flower and leaf extracts, regardless of the solvent used, indicating lower antioxidant effectiveness. Furthermore, when acetone or ethyl acetate is used as the solvent, IC50 values tend to be higher, suggesting that these solvents are less efficient in extracting antioxidants compared to methanol. Differences between the antioxidant activity of extracts obtained by different extraction solvents were significant (p>0.05).

Table 5.
Antioxidant activity of investigated F. ulmaria extracts, IC₅₀ values (µg/mL).

Extract	Methanol	Acetone	Ethyl acetate
Root	113.06±0.01a	177.62±1.14c	161.38±0.53b
Stem	165.82±0.01a	226.27±1.14c	208.05±0.53b
Leaf	28.27±0.53a	64.81±0.01b	146.1±1.14c
Flower	13.27±0.57a	30.65±0.15b	52.1±1.05c

• Values are mean ± standard deviation of triplicate analyses. Values within the row followed by the same letter (a, b, c), are not significantly different according to Tukey’s test (p<0.05).

Antimicrobial activity of the extracts against Gram-positive and Gram-negative bacteria

In this study, the antibacterial activity of the extracts ranged from 0.312 mg/ml to 10 mg/ml. No statistically significant difference in activity among tested plant extracts was noticed (p < 0.05), except between acetone and ethyl acetate extracts (p = 0.023). The values obtained for the antibacterial activity of extracts are presented in Table 6.

Table 6.
Antibacterial activities of the leaves extracts of F. ulmaria

	Methanol extract	Ethyl-acetate extract	Acetone extract	Tetracycline
	MIC (mg/ml)			MIC (µg/ml)
Gram-positive bacteria
Staphylococcus aureus ATCC 25923	0.63	1.25	0.31	1.50
Staphylococcus aureus (isolate)	2.50	5.00	1.25	<0.125
Bacillus cereus (isolate)	0.63	1.25	0.63	0.25
Gram-negative bacteria
Escherichia coli ATCC 25922	10.00	>10	5.00	4.00
Klebsiella pneumoniae ATCC 70063	5.00	>10	5.00	4.00
Pseudomonas aeruginosa ATCC 27853	10.00	10.00	10.00	4.00
Escherichia coli (isolate)	10.00	>10	5.00	2.00
Klebsiella pneumoniae (isolate)	5.00	>10	5.00	4.00
Salmonella enterica (isolate)	5.00	>10	5.00	2.00

Antibacterial activity was detected against all tested Gram-positive strains. The methanol extract showed significant antibacterial activity against Staphylococcus aureus ATCC 25923 (MIC 0.63 mg/ml) and Bacillus cereus (MIC 0.63 mg/ml). Similarly, the acetone extract exhibited significant activity against Staphylococcus aureus ATCC 25923 (MIC 0.31 mg/ml) and Bacillus cereus (MIC 0.63 mg/ml). The ethyl acetate extract displayed slightly weaker but still significant activity against these bacteria, with MIC values of 1.25 mg/ml for both Staphylococcus aureus ATCC 25923 and Bacillus cereus. However, methanol, acetone, and ethyl acetate extracts showed relatively weak activity against Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and others, with generally high MIC values, indicating lower antibacterial effectiveness compared to tetracycline. In summary, the results suggest that methanol and acetone extracts from the leaves of F. ulmaria exhibit significant antibacterial activity against selected Gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) but are less effective against Gram-negative bacteria. The ethyl acetate extract also demonstrates antibacterial activity but is slightly less effective than methanol and acetone extracts. Tetracycline exhibits high activity against most tested bacteria, serving as a potent control agent in this study.

Discussion

The species F. ulmaria is traditionally used for pain relief, treating various inflammatory diseases, digestive system disorders, and liver diseases (Farzaneh et al., 2022). The medicinal properties of F. ulmaria are attributed to numerous phytochemical compounds (flavonoids, tannins, phenolic acids, terpenoids, salicylic acid derivatives, etc.) responsible for its anticancer, antioxidant, antimicrobial, and anti-inflammatory effects, present in both vegetative and reproductive organs (Baranenko et al., 2019; Bijttebier et al., 2019).

A study by Orhan et al. (2007) suggests that the content of phenolic compounds can be influenced by genotype, collection location, cultivation technique, and plant maturity. External factors such as temperature, light, moisture level, the presence of nutrients in the soil, and altitude can affect the phenylpropanoid metabolism of the plant (Popović et al., 2017).

The distribution of pharmacologically active secondary metabolites is uneven throughout the plant. The root of F. ulmaria is poorly researched. Our research shows that the antioxidant activity and the content of phenols and tannins in methanol and acetone extracts of the root are higher than in the corresponding extracts of the stem but slightly lower than in the leaves. This suggests that the root can also be used for medicinal purposes. The methanol extract of the F. ulmaria root exhibits no genotoxic activity (Matić et al., 2015).

The highest quantities of flavonoids were found in the flowers of F. ulmaria. The following flavonoids were isolated from the flowers and leaves: quercetin-4'-O-glucoside, quercetin-3'-glucuronide, quercetin-3-O-arabinoside, quercetin-3-O-galactoside, quercetin-3-O-ramno-glucoside, kaempferol-4'-glucoside, quercetin, quercetin-3-O-glucofuranoside, quercetin-4'-O-galactopyranoside, and quercetin-3-O-glucopyranoside (Krasnov et al., 2006). Kikuchi et al. (2019) suggest that kaempferol, luteolin, and quercetin are responsible for the antiangiogenic and antimetastatic effects of F. ulmaria. Our study also noted that the highest flavonoid concentration was detected in flowers (methanol extract), while the quantity of flavonoids in stem and root extracts was near the detection limit. These findings are consistent with the results of the study by Barros et al. (2011). The comparison of two studies (Barros et al., 2011; Pukalskienė et al., 2015) with our research shows that higher amounts of secondary metabolites (phenols and flavonoids) are detected in samples from Mediterranean climates (Barros et al., 2011), while the lowest concentration levels are observed in samples from temperate climatic areas (Pukalskienė et al., 2015). Lima et al. (2014) assert that flower extracts (methanol and methanol-water) reduce the cell proliferation of human tumor cell lines.

Antioxidant properties are the most important biological activities of plants and plant products justifying their role in various pathophysiological conditions and protection of organs. They are mainly determined by the presence of polyphenolic compounds. Ethanol extracts of the above-ground part of the plant showed strong antioxidant activity (Shilova et al., 2006). Our results show that the root has moderate and the stem low antioxidant activity, which is in line with the results by Savina et al. (2023).

The World Health Organization warns of the continuous rise of pathogens resistant to conventional drugs, leading to over 20,000 deaths in Europe annually due to infections caused by antibiotic-resistant bacteria. For this reason, there is a need to search for potential new sources of antimicrobial agents, especially from plants that have noteworthy traditional medicinal use (Manandhar et al., 2019). The aqueous leaf extract of F. ulmaria exhibited no significant antimicrobial activity noted in studies by Woods-Panzaru et al. (2009). The flower extract of F. ulmaria showed antifungal effects against Candida species (C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis) (Woods-Panzaru et al., 2009). In Katanić's study (2017), extracts of above-ground and underground parts of F. ulmaria showed activity against all tested bacteria with MIC values ranging from 0.156-10 mg/ml. In our study, F. ulmaria exhibited mild antimicrobial activity against Gram-negative bacteria, starting at 10 mg/ml, which also matches Katanić's (2017) research where the value was 5 mg/ml. By comparing the activity against S. aureus strains, our study showed that S. aureus ATCC 25923 was more sensitive than the clinical isolate of S. aureus. In Katanić's study (2017), the opposite was observed revealing better antimicrobial activity against S. aureus isolates compared to the standard strain of S. aureus. The differences in results can be explained due to the different S. aureus isolates used and possible differences in the chemical composition of plants collected at different times and in different geographical areas. MIC values of plant extracts are significantly higher and cannot be easily compared with the values of antibiotic activity obtained for the standard antibiotic (tetracycline). Inhibitory activity of the reference antibiotic tetracycline showed very low minimum concentrations, except for P. mirabilis isolate which showed resistance to tetracycline.

The extract of F. ulmaria could potentially be used as a natural preservative against inoculated pathogenic bacteria in fish meat and fish roe products (tarama salad) (Boziaris et al., 2011).

Based on the analysis of the phytochemical composition and biological activity of extracts from roots, stems, leaves, and flowers of F. ulmaria, we can conclude that flowers and leaves stand out as rich sources of phenols, flavonoids, and total tannins. The methanol extract of the flower exhibits exceptional antioxidant and antibacterial activity. On the other hand, stems and roots have lower concentrations of flavonoids and tannins and lower antioxidant activity, regardless of the solvent used. Methanol and acetone extracts from the leaves of F. ulmaria demonstrate significant antibacterial activity against Gram-positive bacteria, such as Staphylococcus aureus and Bacillus cereus. However, their effectiveness against Gram-negative bacteria, including Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, is relatively low. The ethyl acetate extract possesses antibacterial activity but is less effective compared to methanol and acetone extracts.

The results of the phytochemical composition and biological properties of various parts of the plant F. ulmaria (roots, stems, leaves, and flowers) provide scientific validation for the ethnomedical use of this species. Detailed phytochemical research under both in vitro and in vivo conditions is necessary in order to assess the potential applications of the entire plant or specific plant organs of F. ulmaria in various contexts, including pharmaceutical products and natural remedies.

Acknowledgments

The authors acknowledge the contribution of Nevena Srđević (Faculty of Science, University of Kragujevac). This research was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Agreement No. 451-03-65/2024-03/200122).

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Publication Dates

• Publication in this collection
  22 Nov 2024
• Date of issue
  2024

History

• Received
  12 July 2023
• Accepted
  15 July 2024