In Vitro Study of Biological Activity of Tanacetum vulgare Extracts

Tanacetum vulgare is an herbaceous plant widely used in folk medicine. It is rich in phenolic acids and flavonoids, which have pharmacological and medicinal properties, such as anthelmintic, antispasmodic, tonic, antidiabetic, diuretic, and antihypertensive. This study aimed to confirm the presence of biologically active substances in Tanacetum vulgare and to determine the pharmacological spectrum of biological activity of Tanacetum vulgare extract components. When preparing Tanacetum vulgare extracts, the highest yield was observed when using the maceration method with a mixture of solvents methanol + trifluoroacetic acid (22.65 ± 0.68%). The biologically active substances in Tanacetum vulgare extract samples were determined using high-performance liquid chromatography. Biologically active substances such as luteolin-7-glucoside (550.80 mg/kg), chlorogenic acid (5945.40 mg/kg), and rosmarinic acid (661.31 mg/kg) were identified. Their structures were determined. The experiments have confirmed the antioxidant and antibacterial activities. Secondary metabolites of Tanacetum vulgare extracts have been found to have previously unknown biological activity types; experimental confirmation of their existence will advance phytochemical research and lead to the development of new drugs.


Introduction
The name Tanacetum vulgare L., also known as Common Tansy, is derived from the Greek word "athanasia", which means "immortality", most likely as a result of the fact that the flowers of this plant do not wilt when dried [1]. This perennial herbaceous plant is widely distributed in North America, Europe, Asia, China, Japan, North Korea, and Russia. Tanacetum vulgare L. has been found growing wild in many states of the United States, Europe, and Asia along roadsides, in wastelands, and as a hedge [2,3]. The chemical composition of the plant and its essential oils is affected by its growing environment and climate. It is well described in the literature for many plants [2]. The plants with sectile leaves grow in Corsica (France), Sardinia, and Sicily (Italy), and some consider them to be a separate species (ssp. siculum) [4,5].
T. vulgare is a 50-100 cm tall perennial herbaceous plant. The stem is straight, branched from the middle, furrowed, glabrous, or slightly pubescent. The leaves are alternate, dark The BASs of T. vulgare extracts for bioanalysis were isolated from crude T. vulgare flower extracts. The BAS standard samples (luteolin-7-O-glucoside, chlorogenic acid, and rosmarinic acid) for the comparison and identification of these compounds in extracts were purchased from AG Analytekspert (Moscow, Russia).

Determination of the Antioxidant Activity of BASs from T. vulgare Extracts
The antioxidant activity (AOA) of the BASs from extracts of T. vulgare was studied using three methods: a method based on the ability to trap free radicals DPPH (2,2-diphenyl-1-picrylhydrazyl); a method based on the ability to trap free radicals ABTS (2,2/-azinobis(3-ethylbenzothiazoline-6-sulfonic acid); and a method based on the restoring power upon interaction with the Fe(III)-2.4.6-tripyridyl-s-triazine (FRAP) complex.
DPPH is a stable free radical that reacts with the hydrogen atom released by the substrate. The antioxidant activity was assessed by the presence or absence of a dark purple color of the DPPH solution and absorption bands in the ethanol solution at a wavelength of 517 nm using a CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany).
To prepare a solution of ABTS radicals, an aliquot of a solution of ABTS with a concentration of 7.0 mmol/L and a solution of potassium persulfate with a concentration of 2.45 mmol/L were mixed and incubated in a dark place at 25 • C for 16 h. Then, 20 µL of BASs or a standard sample was added to 300 µL of the prepared solution of the ABTS radical cation. The mixture was incubated for 15 min at 37 • C in the dark and the optical density was determined at 734 nm on a UV-1280 spectrophotometer (Shimadzu, Kyoto, Japan).
The antioxidant activity of the biologically active substances was studied using a FRAP solution. For this, one part of a 10 mmol/L solution of 2,4,6-tripyridyl-s-triazine in an HCl solution with a concentration of 40 mmol/L, one part of a FeCl 3 × 6H 2 O solution with a solution concentration of 20 mmol/L, and 10 parts of an acetate buffer with concentration of 0.3 mol/L (pH = 3.6) were mixed [23]. The reaction was performed at 37 • C in the dark for 10 min. The light absorption was measured at 593 nm on a UV-1280 spectrophotometer (Shimadzu, Kyoto, Japan).

Determination of the Antimicrobial Activity of BASs from T. vulgare Extracts
The antimicrobial properties of biologically active substances were studied using the disk-diffusion method against test strains of Bacillus subtilis (Gram-positive bacterium), Escherichia coli, Pseudomonas aeruginosa (Gram-negative bacteria), Candida albicans (yeast). E. coli, P. aeruginosa, and B. subtilis strains were cultivated on solid and liquid LB nutrient mediums (Dia-M, Moscow, Russia) at 37 • C. The strains of microscopic fungi C. albicans were cultivated on a Ringer's nutrient medium (Dia-M, Moscow, Russia) at a temperature of 25 • C. The following concentrations were used to determine antimicrobial activity: 0.625, 1.25, 2.5, 5, 10, 25, 50, 100, 150, and 200 µg/disk. An antibiotic (kanamycin) at a concentration of 50 µg/disk (for bacteria) and fluconazole at a concentration of 500 µg/disk (for yeast-like fungi-C. albicans) were used as a positive control. A solution of TFA (31%) and acetonitrile (69%) was used as a negative control.
Three parallel measurements were recorded. The measurement result was selected as the mean value.

Molecular Docking
In the Schrödinger software package for selected antibiotic activity target proteins, the molecular modeling of binding to isolated natural compounds was performed. The molecular modeling algorithm was standard and consisted in the preparation of selected target proteins, preparation of ligands, subsequent docking, and evaluation of target binding. The compounds are ranked in scoring function values as Gibbs binding energies. The crystal structures of all the targets were downloaded from RCSB PDB database (https://www. rcsb.org/ accessed on 9 February 2023) with corresponding PDBID-transcription initiation complex (6VVT) [24], dihydrofolate reductase (2WV3) [25], elongation factor G (2BV3) [26], enoyl-acyl carrier protein reductase (2PD4) [27], and deacetylase LpxC (2GO4) [28]. All the ligands, ions, and water molecules were removed from the structures. Hydrogen was first added to each structure, and then the polar hydrogen atoms were removed. The compound library was prepared in the LigPrep module. Molecular docking was performed using the Schrödinger software package (Schrödinger, LLC: New York, NY, USA, 2017) using the gelid algorithm with the corresponding scoring function calculated [29,30].

Statistical Data Processing
The obtained data were statistically processed using the SigmaPlot 12.3 program (Systat Software GmbH, Erkrath, Germany). The obtained indicators were expressed as the mean value ± standard deviation. All the experiments were repeated three times. A one-way ANOVA was used to determine statistically significant differences between the Pharmaceutics 2023, 15, 616 5 of 14 mean values. Using the Shapiro-Wilks test and uniformity of variance, the data were tested for normal distribution before ANOVA analysis. The Tukey test was used to find the differences between the data's mean values at a significance level of p < 0.05.

Results
The maximum yield of dry extracts was determined in the experiment. Table 1 shows the total yield of T. vulgare plant extracts. Since the highest extract yield (22.65 ± 0.68%) was observed when using the method of maceration of T. vulgare with a mixture of methanol and trifluoroacetic acid (TFA). Then, HPLC was used to study the composition of all the obtained extracts ( Table 2). Among the methanolic extracts, the highest yield was observed during extraction using the Soxhlet method; therefore, this extract was subjected to further study. Table 2 lists the BASs identified in the T. vulgare extract samples. The extract producing methanol with trifluoroacetic acid had the highest concentration of the phenolic components. This extract was selected for research on biological activity. All the other identified BASs that are demonstrated in the chromatogram ( Figure 1) were found in trace amounts. In addition to the components listed in Table 2, the chromatogram of this extract showed unidentified peaks of high intensity (24.3 min, 35.7 min, and 41.5 min); the absorption spectra of which corresponded to phenolic acids. Further research is needed to identify these components. The extract producing methanol with trifluoroacetic acid had the highest concentration of the phenolic components. This extract was selected for research on biological activity. All the other identified BASs that are demonstrated in the chromatogram ( Figure  1) were found in trace amounts. In addition to the components listed in Table 2, the chromatogram of this extract showed unidentified peaks of high intensity (24.3 min, 35.7 min, and 41.5 min); the absorption spectra of which corresponded to phenolic acids. Further research is needed to identify these components. The HPLC chromatogram of the T. vulgare methanol extract with 0.1 N TFA is presented in Figure 1.    The HPLC chromatogram of the T. vulgare methanol extract with 0.1 N TFA is presented in Figure 1. tion of the phenolic components. This extract was selected for research on biological ac-tivity. All the other identified BASs that are demonstrated in the chromatogram ( Figure  1) were found in trace amounts. In addition to the components listed in Table 2, the chromatogram of this extract showed unidentified peaks of high intensity (24.3 min, 35.7 min, and 41.5 min); the absorption spectra of which corresponded to phenolic acids. Further research is needed to identify these components. The HPLC chromatogram of the T. vulgare methanol extract with 0.1 N TFA is presented in Figure 1.              The predetermined BAS detection threshold is small (about 20%), which indicates a rare structure of the compound for the predicted activities. We focused on the three main components of T. vulgare extracts in this study: lutelin-7-glucoside, chlorogenic, and rosmarinic acids, because they are found in the greatest quantities in the T. vulgare extracts and have the highest antioxidant and antimicrobial activity when compared to other identified biologically active substances (3,4-dihydroxybenzoic acid, neochlorogenic acid, caftaric acid, caffeic acid, coumaric acid, ferulic acid, and apigenin-7-O-glucoside). Lutelin-7-glucoside, chlorogenic, and rosmarinic acids are well-known compounds with numerous research papers describing their properties [31][32][33].
The simulation results show a high binding capacity of the natural compounds found in the T. vulgare extracts to the selected targets. For each target, natural compounds capable of binding were found. The highest binding properties are: Neochlorogenic acid, Luteolin-7-glucoside, Apigenin-7-O-glucoside, and Chlorogenic acid. The following enzymes have the most sensitive binding: enoyl-acyl carrier protein reductase, which is The predetermined BAS detection threshold is small (about 20%), which indicates a rare structure of the compound for the predicted activities. We focused on the three main components of T. vulgare extracts in this study: lutelin-7-glucoside, chlorogenic, and rosmarinic acids, because they are found in the greatest quantities in the T. vulgare extracts and have the highest antioxidant and antimicrobial activity when compared to other identified biologically active substances (3,4-dihydroxybenzoic acid, neochlorogenic acid, caftaric acid, caffeic acid, coumaric acid, ferulic acid, and apigenin-7-O-glucoside). Lutelin-7-glucoside, chlorogenic, and rosmarinic acids are well-known compounds with numerous research papers describing their properties [31][32][33].
The simulation results show a high binding capacity of the natural compounds found in the T. vulgare extracts to the selected targets. For each target, natural compounds capable of binding were found. The highest binding properties are: Neochlorogenic acid, Luteolin-7glucoside, Apigenin-7-O-glucoside, and Chlorogenic acid. The following enzymes have the most sensitive binding: enoyl-acyl carrier protein reductase, which is prone to the selective action of Luteolin-7-glycoside and Apigenin-7-O-glucoside, and deacetylase LpxC, which selectively binds neochlorogenic, chlorogenic, and cafftaric acids. The high selectivity of binding of caftaric acid to elongation factor G (2BV3) is noticeable. From the modeling results, it can be concluded that the potentiation of the 2PD4 and 2GO4 enzymes can lead to an antibacterial effect against gamma-positive and Gram-negative bacteria. The results of molecular docking are in good agreement with the literature data on the study of the potentiation of the enzymatic targets of our choice using antibacterial drugs with new mechanisms of action [34][35][36]. Table 3 presents the scoring function (Gibbs energy in kcal/mol) to the corresponding compound end target.  Figure 6 depicts the interaction of amino acid side chains of binding sites with most potent ligands and their targets.
The antioxidant and antimicrobial properties of biologically active substances in T. vulgare extracts were studied to confirm the biological activity. The antioxidant activity (AOA) of the BASs from the T. vulgare extracts was assessed using three different methods to produce more convincing results: the capacity to bind free radicals DPPH and ABTS, as well as the reducing power when interacting with the Fe (III) complex (FRAP). Six crude extracts of T. vulgare were obtained for analysis in the first stage, and individual biologically active substances of these extracts were isolated: lutelin-7-glucoside, chlorogenic, and rosmarinic acids, and compared with pure, standard samples of lutelin-7-glucoside, chlorogenic, and rosmarinic acids purchased for analysis. Table 4 presents the results of studying the antioxidant activity of individual biologically active substances isolated from T. vulgare extracts (lutelin-7-glucoside, chlorogenic, and rosmarinic acids).
The results of determining the antimicrobial activity of BASs from T. vulgare extracts are presented in Table 5.  The antioxidant and antimicrobial properties of biologically active substances in T. vulgare extracts were studied to confirm the biological activity. The antioxidant activity (AOA) of the BASs from the T. vulgare extracts was assessed using three different methods to produce more convincing results: the capacity to bind free radicals DPPH and ABTS, as well as the reducing power when interacting with the Fe (III) complex (FRAP). Six crude extracts of T. vulgare were obtained for analysis in the first stage, and individual biologically active substances of these extracts were isolated: lutelin-7-glucoside, albicans Values in row followed by the same letter do not differ significantly (p < 0.05) as assessed using the post hoc test (Duncan's test).
The results show that in vitro antimicrobial activity of 5 µg/disk of luteolin-7-glucoside against B. subtilis, P. aeruginosa, and E. coli and 10 µg/disk of luteolin-7-glucoside against C. albicans; 5 µg/disc of chlorogenic acid exhibited antimicrobial activity against B. subtilis, P. aeruginosa, and E. coli but no antimicrobial activity against C. albicans; 5 µg/disk of rosmarinic acid exhibits antimicrobial activity against B. subtilis, P. aeruginosa, and E. coli, but not against C. albicans.
The data above demonstrated that the wide range of pharmacological effects and mechanisms of action that the BASs from T. vulgare extracts can have was confirmed by the diversity of the biological activities of these BASs. Particularly, several activities indicated that investigations into the potential use of these compounds as pharmaceutical ingredients with previously unknown antimutagenic, antihypoxic, antioxidant, antibacterial, and cardioprotective actions are promising. In the case of synergism or antagonism of the combined action of biologically active substances in real extracts, in vitro and in vivo experiments are required to confirm the presence of the activities [39][40][41].
To study the mechanism of antioxidant and antibacterial properties, Filimonov et al. [39] studied the processes of molecular docking of carvacrol with xanthine oxidase enzyme and antibacterial protein. In the course of the study, the author proved that the xanthine oxidase enzyme is actively involved in the formation of reactive oxygen species, and the carrier protein reductase is a target for inhibiting bacterial growth [39].
In a study [15], extracts of T. vulgare inflorescences were obtained, which were distinguished by their high (8.8 mmol Trolox equivalent/g) antioxidant activity and determined by the value of their DPPH inhibition. The obtained values of the antioxidant activity of the T. vulgare extracts correlate well with the total content of the phenolic acids [11,21]. These results confirm that the presence of chlorogenic and rosmarinic acids affect the antioxidant activity of extracts of T. vulgare inflorescences. The presence of antioxidant activity of T. vulgare plant extracts confirm the high probability of antioxidant activity of individual BASs isolated in our studies. The difference in the values of antioxidant activity obtained in our study and the values of antioxidant activity obtained in the study [15] is explained by different habitats of T. vulgare.
The research by Devrnja et al. [1] enabled it to be possible to establish that the extract of T. vulgare has the ability to inhibit biofilm synthesis and also exhibits antioxidant and antimicrobial properties. The same research group showed the possibility of using T. vulgare extract in the pharmaceutical and cosmetic industries [1].
A study [43] aimed to determine the antibacterial and antioxidant activities of T. vulgare hydroethanol extracts based on their chemical profiles. The dominant compound of T. vulgare extracts, determined using gas chromatography with mass spectrometry, was trans-chrysanthenyl acetate (18.39%). HPLC-DAD was used to determine the chemical composition of phenolic acids and flavonoids. Chicoric acid was the dominant phenolic compound (4311.3 mg × 100 g −1 ). The of T. vulgare extract had a high antioxidant potential (determined using FRAP and DPPH).
The plant extracts have been found to have antioxidant and antimicrobial properties, causing them to be promising raw materials for the pharmaceutical and food industries. These properties were also confirmed in studies by Bączek et al. [43]. They found that extracts of T. vulgare are characterized by antibacterial, fungicidal, and antioxidant properties. The phytochemical analysis of plant extracts revealed the presence of flavonoids (luteolin, quercetin, and apigenin) and phenolic acids (dicaffeoylquinic acid, caffeic acid, and chlorogenic acid) [43].
In a study [44], T. vulgare essential oil extracts demonstrated antioxidant activity, significantly inhibiting tert-butyl hydroperoxide-induced DCFH oxidation. Sharopov et al. [44] proved that thymol, carvacrol, and eugenol, which belong to the terpenoid series, have antioxidant, antimicrobial, and antifungal activity [45]. It was established that the α-pinene and caryophyllene oxide compounds contained in extracts of T. vulgare have the highest antioxidant activity [46]. The paper [47] described moderate levels of α-pinene antioxidant activity based on DPPH analysis. However, in [47], the antioxidant activity of caryophyllene oxide was reported for the first time.
As reported in [48], T. vulgare extracts have antibacterial activity [48][49][50]. The antibacterial activity of T. vulgare from Romania [51] and Slovakia [52] against strains of Gram-positive bacteria using the disk diffusion method has also been reported. The extracts of the essential oil of T. vulgare from Tajikistan were found to be weakly effective against E. coli and MRSA [44]. The studies show that T. vulgare essential oil extracts are effective against S. aureus and E. coli. In addition, the caryophyllene oxide and γ-terpinene extracts were also active against S. aureus [53]. The antibacterial activity of caryophyllene oxide and γ-terpinene from T. vulgare extracts has been previously reported in the literature [54,55].
It has been proven through in vitro experiments that T. vulgare extracts can exhibit a wide range of activities because of the presence of luteolin-7-glucoside, chlorogenic, and rosmarinic acids, but the most likely ones are antimutagenic, antihypoxic, antioxidant, antibacterial, and cardioprotective activities. This study also demonstrated that the dominant components (luteolin-7-glucoside, chlorogenic, and rosmarinic acids) have antioxidant and antimicrobial activity. Previously unknown types of biological activity of T. vulgare extract secondary metabolites have been identified, and experimental confirmation of their presence will contribute to the advancement of phytochemical research and the development of new drugs.