Filipendula vulgaris Moench Extracts: Phytochemical Research and Study of Their Cytotoxic and Antitumour Activity

Abstract

Filipendula vulgaris Moench (syn. F. hexapetala Gilib., dropwort, Rosaceae) is widely used in folk medicine as an antitumour agent, but there is a lack of scientific knowledge about it. This research aimed to study the phytochemical composition and cytotoxic and antitumour activity of aqueous and aqueous–alcoholic extracts from rhizomes with roots of F. vulgaris to investigate their effect on the development of experimental Ehrlich ascites carcinoma in mice, and their effect on the animals’ lifespan. A total of 10 phenolics and 14 amino acids were determined by HPLC in the extracts. The dominant phenolic compounds were procyanidins B1, B2, and C1, as well as metabolites of the tannins (+)-catechin and epicatechin gallate. For the first time, 27 volatile substances were identified and semiquantified using GC-MS. The principal volatile components were palmitic acid (41.0%), methyl salicylate (24.2%), and benzyl salicylate (17.5%). The aqueous–alcoholic extract was significantly more effective than the aqueous one. The treatment of mice with Ehrlich carcinoma using the F. vulgaris aqueous–alcoholic extract normalised the studied indicators. The growth inhibition coefficient of Ehrlich ascites carcinoma was 62.3% and 65.8% on the 7th and 14th days, respectively. This was manifested in the inhibition of tumour growth based on a decrease in the content of ascites fluid in the abdominal cavity; a more intense manifestation of cytotoxic action on cancer cells; improvements in haematopoiesis, the antioxidant defence system, and the content of the studied bioelements in the blood serum; and an increase in the lifespan of experimental animals by around two times. The study results allow us to consider F. vulgaris rhizomes with roots as a promising anticancer agent for the design of pharmaceutical preparations and further study their effects on the human body.

1. Introduction

At the current stage of creating new drugs, more and more attention is paid to medicinal plants. It is important to study medicinal plants that have centuries-old use in scientific and folk medicine and exhibit antitumour activity. Such plants include Filipendula vulgaris Moench (syn. F. hexapetala Gilib., Filipendula Gilib. genus, Rosaceae family), which is native to Europe and north Asia and has been introduced in New York, New Zealand North, New Zealand South, Newfoundland, Primorye, and Vermont [1]. This plant is widely distributed in Ukraine and is used in folk medicine, which allows for industrial harvesting. F. vulgaris is distributed throughout Ukraine, except for the Carpathians, in the steppe and forest–steppe zones. The possibility of plant cultivation has also been investigated [2]. Information on the pharmacological properties of F. vulgaris remains limited, and the study of specific activity, in particular antitumour activity, is important [3,4,5].

As a result of phytochemical studies of the above-ground and underground organs of F. vulgaris using chemical and physicochemical methods of analysis, including various types of chromatography, phenolic compounds such as hydroxycinnamic acids, flavonoids, glycosides, and tannins, as well as amino acids, fatty acids, carbohydrates, organic acids, and macro- and microelements, were identified [6,7,8].

Polyphenols are the main biologically active substances in the raw material of F. vulgaris. They play an important role in the implementation of antioxidant, anti-inflammatory, and antitumour activity [3,4]. The content of flavonoids, such as quercetin and kaempferol and their glycosides, determines the antioxidant potential of the plant [9,10]. Quercetin helps protect cells from oxidative stress and exhibits antiproliferative activity. Kaempferol is known for its anti-inflammatory properties. The concentration of flavonoids in dry raw material varies within 3.5–4.0%, depending on the environmental conditions. In the composition of F. vulgaris, phenolic acids (chlorogenic, caffeic, and ferulic acids) were found to make up about 1.8–2.3%. They can inhibit angiogenesis (the formation of new blood vessels necessary for tumour growth). Chlorogenic acid is one of the main phenolic compounds of F. vulgaris. It has antioxidant, anti-inflammatory, and antitumour properties. Of particular interest to scientists is its ability to inhibit the growth of hepatocarcinoma cells (HepG2), which is one of the most common and aggressive forms of liver cancer. Chlorogenic acid demonstrates potent antiproliferative activity against hepatocarcinoma cells by inducing apoptosis, blocking proliferation, and reducing oxidative stress and angiogenesis [11,12,13]. The tannin fraction of F. vulgaris is represented by hydrolysed and condensed tannins. Their content reaches 6–10% in dry mass. They can exhibit antitumour effects through inhibition of tumour signalling pathways and activation of the immune response, effectively inducing apoptosis in lung cancer cells [14,15,16].

Extracts of F. vulgaris rhizomes with roots also contain triterpene saponins, which can cause lysis of tumour cell membranes and inhibit their proliferation [16,17]. Essential oils additionally contribute to the protection of cells from oncogenic factors [4,14]. The raw material also contains aqueous-soluble polysaccharides (2.5–4.0%), in particular pectins (1.5–2.2%), which regulate the work of the gastrointestinal tract, contributing to the detoxification of the body [18,19]. Glycans have immunostimulatory activity [20]. Given this composition, it can be assumed that rhizomes with roots of F. vulgaris can exhibit antitumour activity through antioxidant action and modulation of cellular metabolism [3,4].

Modern oncology requires new effective agents, in particular those of natural origin, which would have minimal toxicity and could be used in combination therapy. Studies of other species of the genus Filipendula have already demonstrated the promise of such plants in the fight against cancer cells. In particular, F. ulmaria (L.) Maxim. exhibits cytotoxic activity against several types of cancer cells [7,21]. To date, there is a lack of sufficient experimental data on the antitumour activity of F. vulgaris, which makes the study of the cytotoxic and antitumour activity of the F. vulgaris rhizome with root extracts relevant and promising.

In Ukraine, in scientific and folk medicine, a decoction of F. vulgaris rhizomes with roots is used as an anti-inflammatory, astringent, and antitumour agent. The rhizome with roots of F. vulgaris is part of tea mixture No. 1 according to Zdrenko’s prescription (a symptomatic remedy for the treatment of papillomatosis of the urinary bladder and antacid gastritis), as well as for treatment of diseases of the gastrointestinal tract, disease of the cardiovascular system, and oncological diseases [22,23,24,25].

A thorough chemical analysis of F. vulgaris revealed a wide range of biologically active substances that have significant therapeutic potential in the prevention and treatment of cancer. In particular, the high content of flavonoids, phenolic acids, tannins, and saponins provides antioxidant, antimetastatic, and apoptosis-inducing effects [26]. Macro- and microelements contribute to the normalisation of metabolism and increased immunity. Studies have confirmed that F. vulgaris can become the basis for the creation of phytopreparations with antitumour activity. Further in vivo studies will help confirm the effectiveness and safety of F. vulgaris extracts in the treatment of cancer.

This research aimed to study phytochemical composition and cytotoxic and antitumour activity of aqueous and aqueous–alcoholic extracts from rhizomes with roots of F. vulgaris to investigate their effect on the development of experimental Ehrlich ascites carcinoma in mice and their effect on the animals’ lifespan.

2. Materials and Methods

2.1. Plant Raw Materials

Rhizomes with roots of F. vulgaris were harvested from the pharmacopoeial plot of medicinal plants near the building of the Faculty of Pharmacy, Ivano-Frankivsk National Medical University (IFNMU), in the middle of October 2023. Voucher specimens No. 613–615 of F. vulgaris were deposited at the Department of Pharmaceutical Management, Drug Technology, and Pharmacognosy at Ivano-Frankivsk National Medical University. Immediately after collection, the rhizomes with roots were washed with cold water, cut into small pieces (1–1.5 cm), and dried in the shade outdoors, avoiding direct sunlight. A total of around 1.0 kg of dry raw material was obtained.

2.2. Extracts Preparation

To obtain extracts from rhizomes with roots of F. vulgaris, the method of fractional maceration was used, which consisted of repeated extraction (two times) of the initial plant material with separate, changing portions of fresh extractant. Around 100 g of the F. vulgaris rhizomes with roots were extracted with 70% ethanol aqueous solution and purified water, and an aqueous–ethanolic extract, FHE1, and an aqueous extract, FHE2, were prepared. The ratio between the raw material and the extractant was 1:10. For obtaining the aqueous extract, a traditional decoction preparation method was used [26]. The liquid extracts were evaporated with a Buchi B-300 rotary vacuum evaporator (Buchi AG, Flawil, Switzerland) to form soft extracts, which were further freeze-dried (lyophilised) with a Scanvac Coolsafe 55-4 Pro freeze-drier (LaboGene ApS, Allerød, Denmark). Before analyses, the extracts were kept in glass containers in a refrigerator at a temperature of 3–5 °C.

2.3. Phytochemical Research

2.3.1. Analysis of Phenolic Compounds by UPLC-MS/MS

Phenolic compounds in Filipendula extracts were analysed both quantitatively and qualitatively using a UPLC-MS/MS system. Chromatographic separation was performed on an Acquity H-class UPLC chromatograph (Waters, Milford, MA, USA) fitted with a YMC Triart C18 column (dimensions: 100 × 2.0 mm, 1.9 µm). A 1 µL sample of the extracts was injected. The column temperature was consistently kept at 40 °C, and the mobile phase flowed at a rate of 0.5 mL/min. For solvent A, an aqueous solution of 0.1% formic acid was utilised, while solvent B consisted of pure acetonitrile. A gradient elution program was applied under the following conditions: solvent B at 5% from 0 to 1 min, increasing to 30% between 1 and 5 min, followed by a linear rise to 50% from 5 to 7 min. Between 7.5 and 8 min, the column was rinsed using solvent B, and from 8.1 to 10 min, the column was re-equilibrated back to its starting conditions of 5% solvent B. Chemical structure analysis of phenolic compounds was conducted using a Xevo triple quadrupole tandem mass spectrometer (Waters, Milford, MA, USA). Negative electrospray ionisation (ESI) mode was utilised to produce ions for MS/MS analysis. The parameters for MS/MS included a capillary voltage set to −2 kV, nitrogen gas for desolvation heated to 400 °C at a flow rate of 700 L/h, a gas flow rate of 20 L/h, and an ion source temperature maintained at 150 °C. Phenolic compounds were identified qualitatively by comparing their MS/MS spectral data and retention times with those of analytical-grade standards. Quantitative determination was performed using standard dilution methods and linear regression fit models [27,28].

2.3.2. Assay of Amino Acids by UPLC-MS/MS

The analysis of amino acids in Filipendula extracts was performed using an Acquity H-class UPLC system (Waters, Milford, MA, USA) coupled with a Xevo TQD mass spectrometer (Waters, Milford, MA, USA). A 1 µL sample of extracts was injected into a BEH Amide column (150 mm × 2.1 mm, 1.7 µm) from Waters, with the column temperature maintained at 25 °C. The mobile phase was composed of 10 mmol ammonium formate with 0.125% formic acid as eluent A and acetonitrile as eluent B, delivered at a flow rate of 0.6 mL/min. A gradient elution method was applied as follows: 0–1 min with 95% eluent B; 1–3.9 min at 70% B; 3.9–5.1 min at 30% B; 5.1–6.4 min for flushing the column with 70% eluent A; at 6.5 min, the gradient was reset to its initial composition for a total runtime of 10 min. The mass spectrometer was operated under the following conditions: positive electrospray ionisation mode with a voltage of +3.5 kV, a cone voltage of 30 V, desolvation gas at a flow rate of 800 L/h and temperature of 400 °C, while the ion source temperature was maintained at 120 °C. Amino acid identification in the F. vulgaris extracts was achieved by comparing the retention times and MS/MS spectral data with those of analytical-grade standards [29]. Quantitative analysis was conducted using linear regression models derived from the standard dilution method [30,31].

2.3.3. Hydrodistillation and Assay of Volatile Compounds by GC-MS

Volatile compounds from 20.0 g of dried rhizomes with roots of F. vulgaris were extracted by hydrodistillation using 300 mL of purified water, following the essential oil distillation protocol outlined in the European Pharmacopoeia [32].

These compounds were analysed with a gas chromatography–mass spectrometry (GC-MS) system, specifically an Agilent 6890/5973 GC-MS (Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with a mass spectrometric detector (MSD) and Chemstation software. Samples of 1 µL each, dissolved in n-hexane, were injected in split mode (ratio 20:1) at an injector temperature of 280 °C. Helium was utilised as the carrier gas, while separation occurred on an Agilent HP-5MSI (Agilent Technologies, Inc., Santa Clara, CA, USA) column (30 m length, 0.25 mm inner diameter, 0.25 µm film thickness). The flow rate was kept steady at 1 mL/min. The column temperature program began at 50 °C and was held for 2 min, then increased at 4 °C per minute until reaching 280 °C, where it was maintained for 5 min [33].

The mass spectrometer operated in electron ionisation (EI) mode at 70 eV. Mass spectra were recorded across a range of 29–400 m/z, with a solvent delay set to 4 min and a scanning speed of 3.8 scans per second. The data were processed with Agilent Masshunter (https://www.agilent.com.cn/en/promotions/masshunter-mass-spec, accessed on 6 May 2025, Agilent Technologies, Inc., Santa Clara, CA, USA) software, utilising deconvolution algorithms and varied window size factors. Compound identification was performed by comparing the NIST23 library with a match factor of ≥ 90 and retention indices calculated using n-alkanes (C7–C30) [34]. The composition of each compound was calculated from chromatogram peak areas without correction factors. GC-MS spectra of the main components are shown in Figures S1–S5.

2.3.4. Standardisation of the Extracts

Quality control methods for the tincture and the dry extract of Filipendula (meadowsweet) rhizomes with roots were implemented [35]. The identification of salicylic acid and major flavonoids was performed using the TLC methods.

Salicylic Acid. The determination of salicylic acid was carried out using the TLC method with mobile phase ethyl acetate—chloroform (1:9). A total of 100.0 mg of the F. vulgaris extracts was dissolved in 10 mL of 96% ethanol. A 0.01 mL aliquot of the extracts was applied to a TLC silica gel plate. After treatment with a 3% ethanolic solution of ferric chloride (FeCl₃), a violet spot corresponding to the standard of salicylic acid was observed at an R_f value of 0.75.

The quantitative determination of salicylic acid was performed using the HPLC method [32,35]. Samples of 100.0 mg of the extracts were dissolved in 10 mL of 5% hydrochloric acid and heated in a water bath for 1 h. The obtained hydrolysate was evaporated to dryness, dissolved in 10 mL of 96% ethanol, and transferred to a 10 mL volumetric flask. The determination was performed using an Agilent HPLC system (Agilent Technologies, Inc., Santa Clara, CA, USA), column 100 mm × 3.0 mm, Agilent Technologies packing material with a particle size of 3.5 μm, fluorescence detector at 310 nm and 450 nm, temperature of 45 °C, flow rate of 1 mL/min, mobile phase of purified water with 0.1% orthophosphoric acid and 85% acetonitrile, and sample volume of 10 μL [36].

Flavonoids. For the identification of dominant flavonoids, the TLC method was used with mobile phase ethyl acetate–water–formic anhydrous acid–anhydrous acetic acid (72:14:7:7) using standard samples of hyperoside and rutin [32]. After spraying the blade with solutions of aminoethyl ester of diphenylboronic acid, macrogol 400, when viewed in UV light, rutin and hyperoside zones were identified.

The quantitative determination of the total flavonoids (expressed as rutin) was performed by the spectrophotometric method [27,32,37]. Approximately 100.0 mg (accurately weighed) of the extracts was placed in a 50 mL volumetric flask and dissolved in 50 mL of 70% ethanol. A 2 mL aliquot of the extract solution was transferred to a 25 mL volumetric flask, followed by the addition of 10 mL of 70% ethanol, 2 mL of a 2% aluminium chloride (AlCl₃) solution, and three drops of diluted acetic acid. The solution volume was adjusted to the mark with 70% ethanol. After 30 min, the absorbance was measured at 407 nm in a 10 mm cuvette using a Shimadzu UV-1800 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). A reference solution was prepared using 2 mL of the extract solution, three drops of diluted acetic acid, and dilution to volume with 70% ethanol in a 25 mL volumetric flask. Simultaneously, the absorbance of a rutin reference solution, prepared similarly to the test solution, was measured [38].

2.4. Cytotoxic and Antitumour Activity

The obtained extracts of F. vulgaris were tested for cytotoxic activity and antitumour properties. The study of biochemical and haematological blood parameters was carried out at the Centre for Bioelementology of Ivano-Frankivsk National Medical University using the atomic absorption spectrophotometry method (accreditation certificate No. 037/19 valid until 12 June 2024).

The study was conducted on white nonlinear mice grown in the vivarium of IFNMU, which were standardised in physiological and biochemical parameters and were kept under the requirements of sanitary and hygienic standards on a standard diet and in compliance with the principles of humane treatment of laboratory animals. During the experiment, the animals were treated following the International Principles of the European Convention for the Protection of Vertebrate Animals Used for Experiments and Other Scientific Purposes [39,40]. These are proven by the Protocol of the Ethics Committee of IFNMU No. 139/23 dated 16 November 2023.

The results of experimental studies were analysed using the Statistic Soft 7.0 mathematical program package with Student’s t-test in Microsoft Office 365 Excel (Exchange Online (Plan 2) for employees of educational institutions). The difference was considered statistically significant at p < 0.05.

To determine the antitumour effect of drugs of plant origin, the most common method is the contact effect of these drugs on ascites tumour cells to establish a cytotoxic effect [41,42,43].

The cytotoxic effect of aqueous and aqueous–alcoholic extracts from rhizomes with roots of F. vulgaris on Ehrlich ascites cancer cells in mice was studied using the R.A. Schreck method, proposed for the primary selection of substances with antitumour effect [41,42].

For the study, ascitic fluid was taken on the 7th–8th day after inoculation of mice with the Ehrlich tumour. A solution of aqueous–alcoholic (FHE1) and aqueous (FHE2) extracts of F. vulgaris rhizomes with roots was added to the ascitic fluid in an isotonic NaCl solution and placed in a thermostat for three hours. A control experiment was conducted in parallel. After incubation in a thermostat, an eosin solution was added, which stained dead cells red. Living cancer cells remained transparent. After 5–10 min, a drop of the mixture was placed in a Goryaev chamber, and the number of dead cells in 10–20 fields of view was counted. Each experiment was repeated 7 times [41].

After studying the cytotoxic effect of the drugs, we studied their effect on the development of experimental Ehrlich ascites carcinoma in mice and the lifespan of the animals.

Ehrlich ascites carcinoma was inoculated into mice by injecting ascitic fluid (n = 1 × 10⁶ Ehrlich tumour cells) into the abdominal cavity under sterile conditions. Twenty-four hours after vaccination, 0.5 mL of a solution of the aqueous–alcoholic and aqueous extract of rhizomes with roots of F. vulgaris was administered intra-abdominally daily for ten days to separate groups of animals at a dose of 50 mg/kg [41].

The study was conducted on 60 white mice weighing 20–24 g, which were divided into groups:

Group Ia—the main group, implanted with Ehrlich carcinoma, treated with the aqueous–alcoholic extract of F. vulgaris rhizomes with roots (n = 14);

Group IIa—the main group, implanted with Ehrlich carcinoma, treated with the aqueous extract of F. vulgaris rhizomes with roots (n = 14);

Group IIIa—intact animals, normal (n = 10);

Group IV—control, implanted with Ehrlich ascites carcinoma, untreated (n = 14).

The studies were conducted in dynamics, with the determination of indicators on the 7th and 14th day. Mice were euthanised under ether anaesthesia, and ascitic fluid, blood, and carcasses of mice without intestines were taken for research. The volume of ascitic fluid was determined before and after treatment. The content of erythrocytes, leukocytes, haemoglobin, and the activity of the enzymes ceruloplasmin, catalase and iron saturation of transferrin were investigated in the blood. The content of the trace elements Fe, Cu, Zn, and Co was determined in the carcasses of mice [44,45].

The growth inhibition coefficient of Ehrlich ascites carcinoma under the influence of the aqueous–alcohol extract of F. vulgaris rhizomes with roots was evaluated on the 7th and 14th days:

$${\mathrm{K}}_{\mathrm{c}}={\displaystyle \frac{6.9-2.6}{6.9}}100=62.3\% {\mathrm{K}}_{\mathrm{c}}={\displaystyle \frac{11.1-3.8}{11.1}}100=65.8\%$$

(1)

The growth inhibition coefficient under the influence of the aqueous extract of F. vulgaris rhizomes with roots was evaluated on the 7th and 14th days:

$${\mathrm{K}}_{\mathrm{B}}={\displaystyle \frac{6.9-4.2}{6.9}}100=39.1\% {\mathrm{K}}_{\mathrm{B}}={\displaystyle \frac{11.1-7.7}{11.1}}100=30.6\%$$

(2)

In a separate group of mice (n = 30) with implanted Ehrlich carcinoma, the effect of treatment with the aqueous–alcoholic and aqueous extracts of F. vulgaris rhizomes with roots on the survival and life expectancy of animals was studied. Ehrlich ascites cancer was inoculated into mice by injecting 0.2 mL of 7- to 8-day ascites fluid (n = 1 × 10⁶ Ehrlich tumour cells) into the abdominal cavity under sterile conditions. Twenty-four hours after inoculation, 0.5 mL of a solution of alcoholic and aqueous extract of rhizomes with roots of F. vulgaris was administered intra-abdominally daily for 10 days to separate groups of animals at a dose of 50 mg/kg.

3. Results

The obtained dry extracts of F. vulgaris rhizomes with roots (FHE1 and FHE2) were loose powders, bitter, odourless, and ranged in colour from light brown to brownish-reddish depending on the extractant used. The extract yield was 24.70 ± 0.31% for FHE1 (extractant: 70% ethanol) and 23.96 ± 0.24% for FHE2 (extractant: water). In these extracts, 10 phenolics and 14 amino acids were identified and quantified using HPLC methods. The dominant phenolic compounds were procyanidins (B1, B2, and C1) and metabolites of tannins ((+)-catechin and epicatechin gallate). Hydroxycinnamic acids were represented by chlorogenic acid, while flavonoids included rutin and hyperoside. In the studied extracts, arginine, proline, alanine, and asparagine were the dominant amino acids. The results of phytochemical research using UPLC-MS/MS and GC/MS are presented in

Table 1. Phytochemical composition of the Filipendula vulgaris in aqueous–ethanolic (FHE1) and aqueous (FHE2) extracts.

Compound	Content of a Compound, mg/100 g
	FHE1	FHE2
Phenolics
Luteolin 7 rutinoside	1.5 ± 0.2	1.7 ± 0.1
Procyanidin B1	690.7 ± 52.7	313.1 ± 12.2
Procyanidin C1	29.6 ± 2.5	40.8 ± 4.5
(+)-Catechin	273.2 ± 2.4	0
Chlorogenic acid	24.7 ± 1.0	15.6 ± 2.0
Procyanidin B2	34.9 ± 2.8	0
3,4-Dihydroxybenzoic acid	13.6 ± 0.7	0
Rutin	4.7 ± 0.4	5.5 ± 0.9
Hyperoside	3.8 ± 0.1	3.9 ± 0.6
Epicatechin gallate	224.5 ± 13.3	0
Amino acids
Alanine	3.7 ± 0.2	4.4 ± 0.1
Arginine	29.7 ± 1.8	44.4 ± 1.5
Aspartic acid	3.8 ± 0.1	7.4 ± 0.6
Glutamic acid	2.5 ± 0.1	3.0 ± 0.1
Histidine	0.8 ± 0.03	0.8 ± 0.02
Isoleucine	1.6 ± 0.1	1.5 ± 0.1
Leucine	1.1 ± 0.1	1.1 ± 0.1
Lysine	0.3 ± 0.06	0.5 ± 0.01
Methionine	0.1	0.1
Phenylalanine	1.2 ± 0.01	1.3 ± 0.05
Proline	9.2 ± 0.1	8.1 ± 0.6
Serine	1.0 ± 0.07	1.4 ± 0.1
Tyrosine	2.3 ± 0.2	2.4 ± 0.2
Valine	0.8 ± 0.04	0.7 ± 0.04
Content of BAS group, %
Salicylic acid derivates	0.27 ± 0.03	0.16 ± 0.02
Flavonoids	4.76 ± 0.57	3.19 ± 0.42

and Figure 1. Additional data related to the identification of phenolic compounds are presented in Figures S6 and S7 and Table S1.

During alcohol extraction from raw materials, terpenes, and other volatile compounds were also extracted and influenced the overall pharmacological activity of the extracts. Therefore, the volatile fraction of F. vulgaris rhizomes with roots was obtained using hydrodistillation in accordance with the European Pharmacopoeia [32] and was subsequently analysed by GC-MS (

Table 2. Composition (>/=0.1%) of volatile compounds of hydrodistillates from rhizomes with roots of Filipendula vulgaris.

Compound	RI	RI NIST23	Content, % (n = 4)
Hexanal	800	801	0.94 ± 0.027
Ethylbenzene	858	855	0.10 ± 0.002
m-Xylene	866	866	4.18 ± 0.133
Benzaldehyde	958	962	0.58 ± 0.013
2-n-Pentylfuran	991	993	0.22 ± 0.007
Salicylaldehyde	1041	1047	1.44 ± 0.058
Heptanoic acid	1074	1078	0.14 ± 0.004
Nonanal	1104	1104	0.10 ± 0.002
DL-Menthol	1173	1173	0.16 ± 0.006
Methyl salicylate	1196	1192	24.20 ± 1.331
L-Carvone	1244	1245	1.76 ± 0.077
Anethole	1286	1287	0.68 ± 0.020
n-Capric acid	1368	1373	0.40 ± 0.005
L-β-Bourbonene	1387	1384	0.22 ± 0.008
(Z)-Jasmone	1399	1394	0.12 ± 0.003
Caryophyllene	1421	1419	0.26 ± 0.009
(E)-β-Farnesene	1458	1457	0.14 ± 0.004
Lauric acid	1565	1567	0.58 ± 0.001
L-Globulol	1586	1591	0.46 ± 0.020
Viridiflorol	1595	1591	0.50 ± 0.019
Acorenone	1692	1685	0.34 ± 0.010
Myristic acid	1764	1768	0.50 ± 0.018
Hexahydrofarnesyl acetone	1846	1844	0.14 ± 0.004
Verimol K	2048	2053	1.48 ± 0.049
γ-Palmitolactone	2098	2105	0.40 ± 0.005
TOTAL			98.54

RI: Retention index.

and Figure 2).

The composition of the volatile fraction of F. vulgaris rhizomes with roots was analysed for the first time. Using GC-MS, 27 substances distilled from the underground parts were identified and semiquantified. The principal components were palmitic acid (41.0%), methyl salicylate (24.2%), and benzyl salicylate (17.5%).

The cytolytic effect of the studied extracts was estimated based on the percentage of dead cells (Figure 3).

As can be seen from the data presented in

Table 3. Haematopoietic parameters during the progression of Ehrlich ascites carcinoma under the treatment with the Filipendula vulgaris extracts on the 7th and 14th day.

Indicators	Norm (n = 10)	Day 7			Day 14
		The Control (n = 7)	Main Groups		The Control (n = 7)	Main Groups
			Ia (n = 7)	IIa (n = 7)		Ia (n = 7)	IIa (n = 7)
Erythrocytes, T/l	9.82 ± 0.07	7.40 ± 0.07 *	8.95 ± 0.20	7.15 ± 0.16 *	6.03 ± 0.4 *	9.66 ± 0.16	6.28 ± 0.15 *
Leukocytes, G/l	7.40 ± 0.16	14.60 ± 0.57 *	11.80 ± 0.18 *	16.10 ± 0.25 *	16.30 ± 0.52 *	8.50 ± 0.20	16.15 ± 0.41 *
Haemoglobin, G/l	114.0 ± 2.37	105.0 ± 2.93 *	106.0 ± 2.79	104.0 ± 3.83 *	102.0 ± 2.50 *	112.0 ± 1.68	101.0 ± 5.48 *

* p < 0.05—probability of difference of indicators with values to the norm.

, the most pronounced cytotoxic effect on cancer cells was exerted by the aqueous–alcoholic extract of the F. vulgaris rhizomes with roots, which at a dose of 0.50 mg caused 66% death of tumour cells. The aqueous extract at a dose of 0.50 mg/kg caused 48% death of cancer cells.

The established cytotoxic effect on cancer cells of the FHE1 extract necessitated additional experimental studies of the effects of the F. vulgaris aqueous and aqueous–alcoholic extracts on the animals’ lifespan, the development of ascites fluid, and some biochemical parameters.

The effects of the F. vulgaris extracts on the growth of Ehrlich’s ascites carcinoma (volume of ascitic fluid) are presented in Figure 4.

The growth inhibition coefficient of Ehrlich’s ascites carcinoma under the influence of the F. vulgaris aqueous and aqueous–alcoholic extracts was determined on the 7th and 14th days of the experiment. They were 39.1% and 30.6%, respectively, for the aqueous extract FHE1, and 62.3% and 65.8%, respectively, for the aqueous–alcoholic extract FHE2. Thus, the aqueous–alcoholic extract of F. vulgaris rhizomes with roots in the treatment of Ehrlich ascites carcinoma exhibited a more pronounced antitumour effect.

Haematopoiesis indicators in the dynamics of the development of Ehrlich ascites carcinoma during the treatment are presented in Table 3.

Analysis of the results of indicators of erythrocytes, leukocytes, and haemoglobin content (

Table 4. The activity of ceruloplasmin, catalase, and iron saturation of transferrin in the dynamics of rats with Ehrlich carcinoma during treatment with Filipendula vulgaris extracts.

Indicator	Norm (n = 10)	Day 7			Day 14
		Control (n = 7)	Main Groups		Control (n = 7)	Main Groups
			Ia (n = 7)	IIa (n = 7)		Ia (n = 7)	IIa (n = 7)
Ceruloplasmin, conventional units	16.23 ± 0.37	14.58 ± 0.63 *	14.28 ± 0.52 *	11.50 ± 0.51 *	12.56 ± 0.46 *	15.10 ± 0.47	11.28 ± 0.49 *
Transferrin, conventional units	0.18 ± 0.003	0.15 ± 0.004 *	0.16 ± 0.006	0.14 ± 0.004 *	0.14 ± 0.004 *	0.17 ± 0.004	0.14 ± 0.006 *
Catalase, mg H2O2/mL	4.45 ± 0.15	3.45 ± 0.10 *	4.08 ± 0.15	3.58 ± 0.11 *	3.48 ± 0.15 *	4.28 ± 0.14	3.28 ± 0.4 *

* p < 0.05—probability of difference between indicators and normal values.

) shows that in the process of Ehrlich carcinoma growth in mice of the control group, along with an increase in the content of ascites fluid in the peritoneal cavity, a significant decrease in the number of erythrocytes and haemoglobin in the blood was observed on the 7th and 14th days of the study. Meanwhile, the level of leukocytes increased sharply and by the end of the experiment exceeded the norm by 2.2 times.

When treating animals of the Ia main group with the F. vulgaris aqueous–alcoholic extract, normalisation of the content of erythrocytes and haemoglobin was observed already on the 7th day and was maintained at this level until the end of the experiment. The number of leukocytes in the blood normalised only on the 14th day of the study.

The treatment with the F. vulgaris aqueous extract in animals of the IIa main group showed a significantly weaker effect. The studied indicators of the content of erythrocytes, leukocytes, and haemoglobin almost did not differ from the indicators established in the control group and at the end of the experiment did not return to the physiological norm.

The development of Ehrlich ascites carcinoma in the control group of mice caused a significant decrease in the activity of such enzymes as ceruloplasmin, catalase, and iron saturation of transferrin throughout the experiment (Table 4), which indicated a significant depletion of the antioxidant system in the animal body.

Under the influence of treatment with the F. vulgaris aqueous–alcoholic extract in the Ia main group, the activity of catalase and iron saturation of transferrin normalised already on the 7th day of the experiment, while the activity of ceruloplasmin gradually increased and on the 14th day of the study corresponded to the physiological norm.

With intra-abdominal administration of the F. vulgaris aqueous extract to mice of the IIa main group throughout the entire study period, no positive effect was found in reducing the activity of the studied enzymes.

Studies of the trace elements content in the carcasses of mice in the control group indicate that the development of Ehrlich ascites carcinoma caused a significant loss of such trace elements as Fe, Cu, Zn, and Co throughout the experiment (

Table 5. Indicators of trace element content in mouse carcasses in the dynamics of rats with Ehrlich carcinoma development during treatment with Filipendula vulgaris extracts.

Microelement	Norm (n = 10)	Day 7			Day 14
		Control (n = 7)	Ia main (n = 7)	IIa Main, (n = 7)	Control (n = 7)	Ia Main (n = 7)	IIa Main, (n = 7)
Fe, mg/kg	55.38 ± 1.01	45.10 ± 0.88 *	46.50 ± 1.35 *	39.25 ± 0.88 *	41.20 ± 0.77 *	52.14 ± 0.66	36.10 ± 1.00 *
Cu, mg/kg	4.54 ± 0.08	3.92 ± 0.04 *	4.10 ± 0.13 *	3.85 ± 0.01 *	3.60 ± 0.08 *	4.18 ± 0.09	3.65 ± 0.008 *
Zn, mg/kg	11.99 ± 0.38	8.58 ± 0.31 *	10.25 ± 0.56 *	8.08 ± 0.24 *	7.62 ± 0.53 *	10.32 ± 0.28 *	7.80 ± 0.26 *
Co, µg/kg	167.7 ± 4.62	131.45 ± 2.02 *	144.8 ± 3.54 *	144.9 ± 2.43 *	130.2 ± 1.75 *	160.10 ± 2.08	151.4 ± 4.86 *

* p < 0.05—the probability of a difference between indicators from the value to the norm.

).

In a separate group of animals (n = 30) with implanted Ehrlich ascites carcinoma, the effect of treatment with the F. vulgaris aqueous–alcoholic and the aqueous extracts on the survival and life expectancy of experimental animals was studied. The results are presented in

Table 6. Effect of Filipendula vulgaris aqueous–alcohol (FHE1) and aqueous (FHE2) extracts on ascites fluid volume and lifespan of animals with implanted Ehrlich carcinoma.

	Ascitic Fluid Volume, mL
	Control Group		Ia Main		IIa Main
	Day 7	Day 14	Day 7	Day 14	Day 7		Day 14
M	6.9 ± 2.8	11.1 ± 0.53	2.6 ± 0.15	3.8 ± 0.20	4.2 ± 0.22		7.7 ± 0.20
n	7	7	7	7	7		7
p			p < 0.001	p < 0.001	p < 0.05		p < 0.05
Life Expectancy
Investigational Drug		Number of Animals	Observation Results
			Complete Absence of Tumour	Animals Died	Lifespan		Cured
					Days	%
–		10	–	10	14.2 ± 0.84	100	–
FHE1 extract		10	–	10	28.0 ± 1.00	197.2	–
FHE2 extract		10	–	10	20.1 ± 0.92	141.5	–

.

The average life expectancy ratio in animals with implanted Ehrlich carcinoma treated with the aqueous–alcoholic extract increased up to 197.2%, while under the influence of treatment with the aqueous extract, it was 141.5%.

4. Discussion

In both F. vulgaris extracts, rutin and hyperoside were identified by TLC, which corresponded with previous results [6,16,17]. Procyanidins, including B1, B2, and C1 found from F. vulgaris extracts, are naturally occurring polyphenols and promising chemopreventive agents that exhibit beneficial health effects, including anti-inflammatory, antiproliferative, and antitumor activities [46]. Procyanidin B2, one of the main phenolics of the F. vulgaris extracts, was proven to have antiproliferative and apoptotic effects and induced autophagy by modulating the Akt/mTOR signalling pathway, so it may be considered as a potential therapeutic drug for gastric cancer [47]. Many flavonoids have shown anticancer activity by affecting different signalling cascades (the PI3K/Akt/mTOR pathway) [48]. Catechins are reactive oxygen species (ROS) scavengers and metal ion chelators, whereas their indirect antioxidant activities comprise induction of antioxidant enzymes, inhibition of pro-oxidant enzymes, and production of phase II detoxification enzymes and antioxidant enzymes. Oxidative stress and ROS are implicated in ageing and related dysfunctions, such as neurodegenerative disease, cancer, cardiovascular diseases, and diabetes [49]. Epigallocatechin gallate was proven to inhibit the mTOR pathway [50].

For the first time, in F. vulgaris rhizomes with roots, 29 volatile substances were identified and semiquantified using GC-MS. The principal components were palmitic acid (41.0%), methyl salicylate (24.2%), and benzyl salicylate (17.5%). Benzaldehyde has been shown to inhibit multiple signalling pathways in cancer cells, including the PI3K/AKT/mTOR, STAT3, NFκB, and ERK pathways 1 [51]. Derivatives of salicylaldehyde have shown potent activity against leukemic cell lines and breast cancer cells [52]. Menthol has demonstrated anticancer activity by inducing apoptosis, causing cell cycle arrest, and inhibiting tumour angiogenesis [53]. Derivatives of methyl salicylate have shown potential as protein tyrosine phosphatase 1B inhibitors with significant anticancer activity [54].

The development of malignant tumours is very common in our time. Long-term chemotherapy against cancer is dangerous for patients, which limits its use in therapy. Therefore, the search for and development of new drugs with antitumour, antimetastatic, and cytotoxic activity while reducing side effects remains relevant [55]. Species of the genus Filipendula have a high content of flavonoids and tannins, as well as phenolic compounds. Extracts of F. ulmaria L. Maxim. and F. hexapetala L. are used in folk and scientific medicine for various diseases, including the treatment of tumours [21,56]. F. ulmaria L. Maxim. can exhibit antitumour activity in colorectal carcinogenesis induced by methylnitrosourea in rats [21]. These studies confirm the relevance of the use of species of the genus Filipendula in oncology and their further in-depth study.

One of the most common tumours is Ehrlich’s ascites carcinoma; therefore, the experimental study of modelling this tumour is of great importance for medicine and pharmacy. Ehrlich ascites carcinoma is a hyperdiploid tumour that develops rapidly, is transplantable, and eventually transforms into an ascites form. The search for safe, effective drugs of natural origin was important for humanity in cancer therapy [41,42]. Several scientists have established the effectiveness of plant extracts in the therapy of Ehrlich ascites carcinoma [43,44,45].

Considering the cytotoxic effect of the extracts (Figure 4), it was found that the aqueous–alcoholic extract of F. vulgaris caused death in 66% of tumour cells at a dose of 50 mg, which significantly exceeded the effectiveness of the aqueous extract (48%). This result can be explained by better extraction of such biologically active compounds as flavonoids, phenolic acids, and tannins, which promote apoptosis of cancer cells, precisely in the aqueous–alcoholic medium. The high cytotoxicity observed here correlated with the results of other studies demonstrating the effectiveness of alcohol extracts in combating tumour cells.

Considering inhibition of the Ehrlich ascites carcinoma growth, on the 7th and 14th day of the experiment, the aqueous–alcoholic extract of F. vulgaris demonstrated higher tumour growth inhibition coefficients (62.3% and 65.8%, respectively) than the aqueous extract (39.1% and 30.6%). These data confirm that the F. vulgaris aqueous–alcoholic extract had a more potent antitumour effect, probably due to the combined effect of antioxidant and cytotoxic components.

A limitation of the study was the absence of normal cells in toxicity and safety studies of F. vulgaris extracts. These are necessary to include in order to understand that the extracts under study are not cytotoxic to normal cells, acting selectively on cancer cell development. Therefore, the normal cells should be included in further, more detailed studies, where it would be appropriate to also consider the possible effects of more apolar extracts.

Studying an effect on haematopoiesis (Table 3), it was found that in the control group, a significant decrease in the number of erythrocytes and haemoglobin was observed, as well as an increase in the level of leukocytes, which is typical for oncological processes because of the influence of tumour intoxication and impaired bone marrow function. Treatment with the F. vulgaris aqueous–alcoholic extract contributed to the normalization of these indicators already on the 7th day of the experiment, which indicates the ability of the extract to stimulate haematopoiesis and reduce the toxicity of the tumour process. At the same time, the aqueous extract had a less pronounced effect on the levels of erythrocytes, leukocytes, and haemoglobin, which indicates its lower effectiveness in regulating haematopoiesis, probably due to insufficient concentration of active components. In general, the positive effects of plant extracts on haematopoiesis have not been demonstrated in many cases. For example, results have supported the haematopoietic potential of Aspilia africana, particularly at moderate doses [57].

The activity of enzymes of the antioxidant system also changed during the experiment (Table 4). The development of Ehrlich ascites carcinoma was accompanied by a decrease in the activity of ceruloplasmin, catalase, and transferrin, which indicates the depletion of antioxidant protection and the activation of free radical processes. Treatment with the F. vulgaris aqueous–alcoholic extract led to the normalization of catalase and transferrin activity on the 7th day, while ceruloplasmin reached physiological values on the 14th day. This demonstrated the antioxidant activity of the extract, which may be associated with the content of phenolic compounds and flavonoids. At the same time, the aqueous extract did not have a pronounced effect on the activity of enzymes, which confirmed its lower therapeutic value compared with the aqueous–alcoholic extract. A link between antioxidant and antitumor effects has also been shown for other plant extracts [58].

The animal body is characterized by an appropriate degree of saturation of tissues and organs with trace metal elements, which play the role of basic modulators for the synthesis of organic structures and biologically active substances, maintaining the stability of homeostasis in a healthy organism. The peculiarity of the biological action of trace elements as biotics is that they activate most enzymatic systems in the tissues of the body; stimulate the processes of tissue respiration, energy metabolism, haematopoiesis, immunological reactions, synthesis of biologically active substances, metabolism of nucleic acids, proteins, carbohydrates, lipids; and adjust the level of free radical processes, depending on the changes that occur in the quantitative content of trace elements in the tissues and organs of the body [59,60,61,62].

In stressful situations, especially those accompanied by malignant growth, corresponding shifts in the redistribution of trace metal elements in tissues occur so quickly that the body does not have time to adapt to changes in metabolic processes, which they correct, contributing to the accumulation of biologically active products that are not specific during normal life, causing an increase in endotoxicosis to a level incompatible with life. The oncological process in the control group was accompanied by significant losses of Fe, Cu, Zn, and Co, which are associated with increased free radical processes, suppression of the antioxidant system, and impaired metabolism of bioelements [63,64].

Treatment with the F. vulgaris aqueous–alcoholic extract contributed to the partial normalisation of the levels of these elements, especially iron and copper, which could be explained by the increase in the activity of ceruloplasmin and transferrin. The use of the F. vulgaris aqueous extract did not affect the progressive decrease in iron content, while the aqueous–alcoholic extract retained iron in the body of mice. On the 14th day of the experiment, iron content corresponded to the physiological norm and amounted to 52.14 ± 0.66 mg/kg (p > 0.05). Iron deficiency in the body of mice, under the influence of malignant growth, is an important pathogenetic indicator that causes a decrease in oxygen delivery to cells and contributes to the inhibition of the synthesis of iron-binding proteins, in particular transferrin, and suppression of the immune status of the body. The latter is accompanied by inhibition of the synthesis of lymphocytoma antibodies, a decrease in the phagocytic function of leukocytes, and a significant impact on the quantity and quality of humoral indicators of natural and acquired immunity: opsonins, precipitins, agglutinins, complement-binding antibodies, and antioxidants [56,62,65,66,67].

Copper as a bioelement is a part of the enzyme ceruloplasmin, which stimulates the processes of haemoglobin formation by increasing iron utilization [59,68]. The significant decrease in copper concentration in the bodies of mice in the control group and the low level of the copper-containing enzyme ceruloplasmin that we established were insufficient to ensure the complete saturation of transferrin with iron, which contributed to the activation of free radical processes against the background of intensification of malignant growth [65,68]. The F. vulgaris aqueous extract did not show a positive effect on the progressive development of copper deficiency in the body. The use of the aqueous–alcoholic extract caused a gradual increase in the concentration of this element to 4.18 ± 0.09 mg/kg, but it did not return to the physiological norm.

Zinc and cobalt, although not reaching the physiological norm, also showed a tendency to increase. At the same time, the aqueous extract did not have a significant effect on the dynamics of the levels of these bioelements, which confirmed its lower effectiveness. Zinc is the main bioelement that regulates the activity of the immune system, modulates the production of cytokines, and stabilises the formation of antioxidant status in the body [55]. Therefore, the decrease in zinc content in the bodies of mice in the control group against the background of the development of Ehrlich’s ascites carcinoma, which we established, is considered as one of the pathogenetic mechanisms that contribute to the weakening of both humoral and cellular immunity against the background of intensification of malignant growth [59,60]. The use of the aqueous extract in mice did not have a positive effect on the gradual increase in zinc deficiency in the body, the concentration of which on the 14th day of the experiment was 7.80 ± 0.26 mg/kg, with a norm of 11.99 ± 0.38 mg/kg. Under the influence of the aqueous–alcoholic extract in the Ia main group, a gradual increase in the zinc content in the carcasses of mice was observed to 10.32 ± 0.28 mg/kg, but it did not return to the physiological norm.

Cobalt as a bioelement plays an important role in the processes of haematopoiesis by enhancing the ionisation and resorption of iron, with its subsequent incorporation into the haemoglobin molecule. In the carcasses of control group mice with implanted Ehrlich carcinoma, we found a significant decrease in this bioelement on the 14th day of the experiment, which was 130.2 ± 1.75 μg/kg, with a norm of 167.7 ± 4.62 μg/kg. Treatment with the F. vulgaris aqueous extract caused a gradual increase in its content by the end of the experiment to 151.4 ± 4.86 μg/kg, but this did not reach the physiological norm. Under the influence of the aqueous–alcoholic extract, the concentration of cobalt normalised.

Thus, the results of the observations presented indicate that the development of experimental Ehrlich ascites carcinoma was accompanied by a decrease in the content of erythrocytes and haemoglobin, and an increase in the number of leukocytes. In addition, there were significant decreases in the activities of ceruloplasmin, catalase, and iron saturation of transferrin in the blood and significant losses of the content of the trace elements Fe, Cu, Zn, and Co in the carcasses of mice. These data indicate profound disturbances in the processes of haematopoiesis, which are caused by a sharp disruption of the homeostasis of trace elements in the animal body due to iron and copper deficiency. This was confirmed by the conducted studies and pronounced decreases in the activity of the copper- and iron-dependent metalloproteins ceruloplasmin and catalase and in the iron saturation of transferrin. The latest data also indicated a profound depletion of antioxidant protection in the body of experimental animals.

The coefficient of average life expectancy in mice treated with the F. vulgaris aqueous–alcoholic extract increased to 197.2%, which significantly exceeded the corresponding indicator for the aqueous extract (141.5%). This confirms that the F. vulgaris aqueous–alcoholic extract was more effective in reducing the toxic effects of the tumour process, reducing the level of intoxication, and improving the general condition of the body.

5. Conclusions

Research on the extracts of F. vulgaris rhizome with roots revealed the promising use of this plant raw material in oncology due to its cytotoxic and antitumour activity. The obtained results indicated the pronounced antitumour potential of the aqueous–alcoholic extract of F. vulgaris, which was confirmed by data on cytotoxic action, effect on haematopoiesis, the activity of enzymes of the antioxidant system, and the levels of bioelements in the bodies of mice with Ehrlich ascites carcinoma. The effects of the underground parts of F. vulgaris, including antitumor activity, were studied for the first time.

In the extracts of F. vulgaris rhizome with roots, a total of 10 phenolics, 14 amino acids, and, for the first time, 27 volatile substances were determined in the underground parts. The main compounds were palmitic acid, methyl salicylate, and benzyl salicylate.

The F. vulgaris aqueous–alcoholic extract had significantly greater antitumour activity than the aqueous extract. Its administration contributed to the inhibition of tumour growth, normalisation of haematopoiesis, restoration of antioxidant enzyme activity, and partial normalisation of the level of trace elements in the body. Thus, the aqueous–alcoholic extract has prospects as a basis for the development of effective antitumour agents of plant origin. However, further studies are needed to clarify the mechanisms of their action and assess their safety.