Beyond Traditional Use of Alchemilla vulgaris: Genoprotective and Antitumor Activity In Vitro

Alchemilla vulgaris L. (lady’s mantle) was used for centuries in Europe and Balkan countries for treatments of numerous conditions and diseases of the reproductive system, yet some of the biological activities of lady’s mantle have been poorly studied and neglected. The present study aimed to estimate the potential of A. vulgaris ethanolic extract from Southeast Serbia to prevent and suppress tumor development in vitro, validated by antioxidant, genoprotective, and cytotoxic properties. A total of 45 compounds were detected by UHPLC–HRMS analysis in A. vulgaris ethanolic extract. Measurement of antioxidant activity revealed the significant potential of the tested extract to scavenge free radicals. In addition, the analysis of micronuclei showed an in vitro protective effect on chromosome aberrations in peripheral human lymphocytes. A. vulgaris extract strongly suppressed the growth of human cell lines derived from different types of tumors (MCF-7, A375, A549, and HCT116). The observed antitumor effect is realized through the blockade of cell division, caspase-dependent apoptosis, and autophagic cell death. Our study has shown that Alchemilla vulgaris L. is a valuable source of bioactive compounds able to protect the subcellular structure from damage, thus preventing tumorigenesis as well as suppressing tumor cell growth.


Introduction
Ethnopharmacological data are of crucial importance for the finding of new promising bioactive compounds, as well as for the verification of already accepted herbal drugs. Therefore, it is necessary to preserve the traditional knowledge of medicinal plants in addition to addressing the need for their sustainable collection from the wild. Nowadays, therapeutic approaches have switched from attacking and directly destroying the damaged cells and pathogenic microorganisms towards the activation of self-healing and protective processes upon the initiation of different repair mechanisms of the human body. Such a view has a great impact on scientific research focusing on the bioactivity of natural products [1]. Numerous pathological conditions cannot be fully treated by standard pharmaceutics [2],

Phytochemical Characterization of A. vulgaris Ethanolic Extract-UHPLC − HRMS
The ethanolic extract of A. vulgaris was investigated by UHPLC-HRMS. A total of 45 compounds (Table 1) were tentatively characterized based on their chromatographic behavior parameters such as retention time, m/z values, molecular formula, error, and fragmentation pattern and comparison with those described in the literature and open access LC-MS libraries. The identified compounds belong to different metabolic classes, mainly phenolics. The values in bold correspond to the base peak.
Compounds 12 and 17-19 were identified as myricetin and gossypetin glycosides, respectively, by comparing their retention times and mass spectral behavior with those published in the literature [28].  [21,24,28]. All these compounds are described for the first time in A. vulgaris. However, the presence of gossypetin glycosides in the studied extract was not very surprising as gossypetin derivatives were isolated from A. mollis [34]. It is worth mentioning that myricetin derivatives have not been detected in Alchemilla species so far.
Compound 30 showed [M − H] − at m/z 447. The MS/MS spectrum presented the characteristic fragmentation patterns for luteolin (m/z 285 and 199) [22]. Therefore, compound 30 was identified as luteolin 7-O-glucoside. This compound has been already described as a component of A. vulgaris [11].  [26]. Compound 38 is reported now for the first time in A. vulgaris, although chrysoeriol has been previously detected in A. vulgaris [15].

Phenolic Acids and Their Derivatives
Gallic acid (1), chlorogenic acid (3), caffeic acid (7), syringic acid (11), p-coumaric acid (13), and p-coumaroylquinic acids (10 and 15) were identified in the studied A. vulgaris extract. The spectra generated for these compounds in negative ion mode gave the de- as well as a typical fragmentation pattern for ellagic acid. Therefore, these compounds were tentatively identified as ellagic acid-hexose and ellagic acid-pentose [23].
Compounds 14 and 5 showed identical fragmentation patterns. Compound 14 with a molecular formula of C 12 H 8 O 6 and a protonated molecule [M − H] − at m/z 247 generated product ions at m/z 219 and 191, resulting from the successive loss of a CO unit. Similarly, in the case of compound 5, the major product ion at m/z 247 was formed via decarboxylation of its precursor ion at m/z 291. The further fragmentation was consistent with that mentioned above for compound 14. According to the literature data, compounds 14 and 5 were identified as brevifolin and brevifolincarboxylic acid [20,25]. Moreover, brevifolincarboxylic acid has been recently described as a component of A. viridiflora [25].

Total Phenolics and Flavonoids and Antioxidant Activity
The results presented in Table 2 represent the results of total phenolic and flavonoid contents and antioxidant capacity. Results for TPC are in accordance with results obtained by Vlaisavljevic et al. [15]. According to Boroja et al., results for TAC and DPPH were lower than those in the current study, which might be a consequence of different solvents, extraction procedures, and the used standard chemicals in performed assays [39]. However, our results are in line with other studies on the antioxidant capacity of the Alchemilla extracts [7,34,40], all indicating the strong correlation between high phenolic content and antioxidant activity, especially concerning a high share of the total flavonoids and tannins in the plant extract [15,41].

Genoprotective Effect of A. vulgaris Extract
Three different concentrations of A. vulgaris extract were tested in vitro for protective effect on chromosome aberrations in peripheral human lymphocytes using a CBMN assay: 2.0 µg/mL, 4.0 µg/mL, and 6.0 µg/mL. The frequency and distribution of MN were scored. The formation of MN after treatment with an alkylating agent, mitomycin C (MMC), and the prevention of MN formation after treatment with DNA repair system agent amifostine WR-2721 were determined. The test system was a peripheral human blood lymphocyte assay, verifying the clastogenic or anticlastogenic effects [42,43]. The possible clastogenic, anticlastogenic, or modulating effects of investigated compounds were determined based on the action of these two agents. The results are presented in Table 3.
Lymphocyte cell culture treated with 1 µg/mL of amifostine WR-2721 showed a significant decrease (p < 0.01) of 18.6% in the frequency of MN compared to control cell cultures (Table 3, Figure 1). Treatment with an MMC alkylating agent at the concentration of 0.2 µg/mL showed a significant increase (p < 0.01) in MN frequency of 24.2% compared to control cell cultures (Table 3, Figure 1). Lymphocyte cell culture treated with 1 µg/mL of amifostine WR-2721 showed a significant decrease (p < 0.01) of 18.6% in the frequency of MN compared to control cell cultures (Table 3, Figure 1). Treatment with an MMC alkylating agent at the concentration of 0.2 µg/mL showed a significant increase (p < 0.01) in MN frequency of 24.2% compared to control cell cultures (Table 3, Figure 1). The statistical significance of the difference between the data pairs was evaluated by analysis of variance (one-way ANOVA) followed by the Tukey test. Statistical difference was considered significant at p < 0.01. a: Compared with control groups, statistically significant difference p < 0.01. b: Compared with amifostine-WR 2721, statistically significant difference p < 0.01. c: Compared with mitomycin C, statistically significant difference p < 0.01.
Three different concentrations of A. vulgaris extract were tested for in vitro protective effect on chromosome aberrations in peripheral human lymphocytes using cytochalasin-B-blocked MN assay. A. vulgaris extract at concentrations of 2.0 µg/mL, 4.0 µg/mL, and 6.0 µg/mL caused a slight decrease in the MN frequency by 20.5%, 18.2%, and 16.3%, respectively, when compared to the control cell cultures (Table 3, Figure 1). Most importantly, A. vulgaris extract at the concentration of 2.0 µg/mL still had a higher protective effect than the synthetic protector, amifostine WR-2721, at the concentration of 1.0 µg/mL (Table 3, Figure 1).
The effect of different concentrations of A. vulgaris extract on cell proliferation was investigated by determination of the cytokinesis-block proliferation index (CBPI). Table 3 shows mean CBPI values and standard errors calculated for different concentrations of A. vulgaris extract. The comparable CBPI values of extracts and amifostine WR-2721 control suggest an inhibitory effect of the tested extracts on lymphocyte proliferation. In this study, we found that the lower concentrations of A. vulgaris extract possess a beneficial effect on lymphocyte cell culture by decreasing the frequency of MN. Since the number of micronuclei serves as an indicator of DNA damage, these results indicate that A. vulgaris extract protects DNA and decreases lipid peroxidation of lymphocytes mostly induced by The statistical significance of the difference between the data pairs was evaluated by analysis of variance (one-way ANOVA) followed by the Tukey test. Statistical difference was considered significant at p < 0.01. a: Compared with control groups, statistically significant difference p < 0.01. b: Compared with amifostine-WR 2721, statistically significant difference p < 0.01. c: Compared with mitomycin C, statistically significant difference p < 0.01.
Three different concentrations of A. vulgaris extract were tested for in vitro protective effect on chromosome aberrations in peripheral human lymphocytes using cytochalasin-B-blocked MN assay. A. vulgaris extract at concentrations of 2.0 µg/mL, 4.0 µg/mL, and 6.0 µg/mL caused a slight decrease in the MN frequency by 20.5%, 18.2%, and 16.3%, respectively, when compared to the control cell cultures (Table 3, Figure 1). Most importantly, A. vulgaris extract at the concentration of 2.0 µg/mL still had a higher protective effect than the synthetic protector, amifostine WR-2721, at the concentration of 1.0 µg/mL (Table 3, Figure 1).
The effect of different concentrations of A. vulgaris extract on cell proliferation was investigated by determination of the cytokinesis-block proliferation index (CBPI). Table 3 shows mean CBPI values and standard errors calculated for different concentrations of A. vulgaris extract. The comparable CBPI values of extracts and amifostine WR-2721 control suggest an inhibitory effect of the tested extracts on lymphocyte proliferation. In this study, we found that the lower concentrations of A. vulgaris extract possess a beneficial effect on lymphocyte cell culture by decreasing the frequency of MN. Since the number of micronuclei serves as an indicator of DNA damage, these results indicate that A. vulgaris extract protects DNA and decreases lipid peroxidation of lymphocytes mostly induced by superoxide anion radicals. The free radicals disturb cellular homeostasis by peroxidation of membrane lipids, oxidation of proteins, base damage, and adduct formation in DNA, which ultimately leads to cell death if the damage is beyond cell repair capacity [44][45][46].

Antitumor Property of A. vulgaris Extract
Human hormone-dependent breast cancer MCF-7, anaplastic melanoma A375, lung adenocarcinoma A549, and colon carcinoma HCT116 cell lines were exposed to a wide range of concentrations of A. vulgaris ethanolic extract, and after 72 h of incubation, cell viability was determined by the measurement of mitochondrial respiration or protein synthesis, using MTT and SRB tests, respectively. As shown in Figure 2, data obtained in both assays confirmed a strong dose-dependent viability decrease in all tested cultures exposed to A. vulgaris extract, apart from malignant cells' origin and characteristics. Since the viability of primary peritoneal exudate cells isolated from healthy animals was not affected by the same range of doses under a comparable experimental setting (Figure 3), it can be concluded that A. vulgaris extract displayed selectivity for the malignant phenotype that is even independent of the hormonal status as believed previously [15]. IC 50 values of all cell lines (Table 4) illustrated the highest effectiveness of A. vulgaris extract against hormone-independent A549 and HCT116 cells. superoxide anion radicals. The free radicals disturb cellular homeostasis by peroxidation of membrane lipids, oxidation of proteins, base damage, and adduct formation in DNA, which ultimately leads to cell death if the damage is beyond cell repair capacity [44][45][46].

Antitumor Property of A. vulgaris Extract
Human hormone-dependent breast cancer MCF-7, anaplastic melanoma A375, lung adenocarcinoma A549, and colon carcinoma HCT116 cell lines were exposed to a wide range of concentrations of A. vulgaris ethanolic extract, and after 72 h of incubation, cell viability was determined by the measurement of mitochondrial respiration or protein synthesis, using MTT and SRB tests, respectively. As shown in Figure 2, data obtained in both assays confirmed a strong dose-dependent viability decrease in all tested cultures exposed to A. vulgaris extract, apart from malignant cells' origin and characteristics. Since the viability of primary peritoneal exudate cells isolated from healthy animals was not affected by the same range of doses under a comparable experimental setting (Figure 3), it can be concluded that A. vulgaris extract displayed selectivity for the malignant phenotype that is even independent of the hormonal status as believed previously [15]. IC50 values of all cell lines (Table 4) illustrated the highest effectiveness of A. vulgaris extract against hormone-independent A549 and HCT116 cells.    To define a precise mechanism beyond the effect of A. vulgaris extract on cell viability, a flow cytometric analysis of cell death was performed, using the A549 cell line as a representative. Namely, after 72 h of incubation in the presence of an IC 50 dose of A. vulgaris extract, a significant amount of early and late apoptotic cells was detected ( Figure 4A). The presence of apoptosis upon the treatment was further confirmed on a morphological level, using DAPI staining of cellular nuclei. Numerous cells with abnormally shaped nuclei and condensed chromatin were visible in cultures exposed to A. vulgaris extract ( Figure 4B). In concordance with a significant presence of apoptotic cells, amplification of total caspase activity in A. vulgaris extract-treated cultures was detected ( Figure 4C). On the other hand, the proliferation of survived cells was abrogated, confirming that the extract suppresses cell division as well ( Figure 4D). Interestingly, a significant amount of autophagosomes was detected in cells cultivated in the presence of A. vulgaris extract ( Figure 4E), while the prevention of autophagosome formation in concomitant treatment with inhibitor 3-MA dramatically restored cellular viability ( Figure 4F). The obtained result clearly confirmed that the intensified autophagic process triggered by the treatment represented an important part of the cytotoxic activity of the A. vulgaris extract.
To exclude the possibility that oxidative stress mediated the antitumor action of A. vulgaris extract, the production of hydrogen peroxide and peroxynitrite was estimated by DHR redox-sensitive dye. However, exposure to the A. vulgaris extract only slightly inhibited the production of ROS and RNS, indicating their insignificance in triggering the apoptotic process ( Figure 5). In summary, the antitumor activity of A. vulgaris extract could be ascribed to inhibited proliferation and both caspase-dependent apoptotic and autophagic cell death.  Detailed analysis of the extract content revealed the presence of numerous biologically active compounds with already recognized antitumor potential. Flavonoids belong to a rich group of polyphenolic compounds in the plant kingdom. Numerous data confirm their strong impact on human health, bringing them into the focus of different scientific studies. The members of the flavonol subclass, quercetin, rutin, and isoquercetin, either as glycosides or aglycones, showed antioxidant, antiproliferative, anti-inflammatory, antihypertensive, and antidiabetic effects. The specificity of naturally occurring compounds is in their high adaptability reflected in support of healthy tissues and healing of pathological conditions. This dual potential of certain extracts or separate compounds is often unexplainable, while the mechanisms triggered by them can be even opposite in different tissues and cells dependent on the platform on which they arrived. Therefore, isoquercetin prevented lipid peroxidation through interference with xanthine oxidase activity, chelation of redox-active metals, or direct scavenging of ROS, exhibiting protective features. On the other hand, the same compound affected the signaling pathways involved in tumor progression, such as the Wnt signaling pathway and mitogen-activated protein kinase, directly affecting tumor viability [47]. Similarly, quercetin, apart from its strong cytoprotective abilities, alters cell cycle progression in neoplastic cells, inhibiting their proliferation, inducing programmed cell death types I and II, and blocking metastasis [48]. Importantly, some herbal compounds possess a highly selective potential and act as Detailed analysis of the extract content revealed the presence of numerous biologically active compounds with already recognized antitumor potential. Flavonoids belong to a rich group of polyphenolic compounds in the plant kingdom. Numerous data confirm their strong impact on human health, bringing them into the focus of different scientific studies. The members of the flavonol subclass, quercetin, rutin, and isoquercetin, either as glycosides or aglycones, showed antioxidant, antiproliferative, anti-inflammatory, antihypertensive, and antidiabetic effects. The specificity of naturally occurring compounds is in their high adaptability reflected in support of healthy tissues and healing of pathological conditions. This dual potential of certain extracts or separate compounds is often unexplainable, while the mechanisms triggered by them can be even opposite in different tissues and cells dependent on the platform on which they arrived. Therefore, isoquercetin prevented lipid peroxidation through interference with xanthine oxidase activity, chelation of redox-active metals, or direct scavenging of ROS, exhibiting protective features. On the other hand, the same compound affected the signaling pathways involved in tumor progression, such as the Wnt signaling pathway and mitogen-activated protein kinase, directly affecting tumor viability [47]. Similarly, quercetin, apart from its strong cytoprotective abilities, alters cell cycle progression in neoplastic cells, inhibiting their proliferation, inducing programmed cell death types I and II, and blocking metastasis [48]. Importantly, some herbal compounds possess a highly selective potential and act as targeted therapy, affecting certain signaling pathways or molecules important for malignant phenotype maintenance. For example, gallic acid functions as an EGFR antagonist, suppressing EGFR-positive NSCLC progression under certain conditions [49]. In addition, this and similar plantderived compounds interfered with chemotherapy, enhancing its effectiveness by changing the pro/antiapoptotic molecule ratio [50].
Apart from mentioned quercetin, isoquercetin, and gallic acid, several other constituents that are present in A. vulgaris extract might also be beneficial in neoplastic conditions since each of them possesses the potential to directly or indirectly influence disease progression per se and in interference with other compounds in the extract. Experience collected from ethnobotanical data and clinical practice confirms that total herbal extracts usually exert more powerful effects than separate compounds. Moreover, some of them, such as quinic acid, have the intrinsic potential to "recognize" selectin-upregulated tumors and induce a transient increase in endothelial permeability to translocate across the endothelial layer. This will result in achieving greater tumor accumulation and delivery of numerous compounds with the direct potential to suppress tumor cell division or viability [51].
The described phenomenon at least partly explains why such compounds can work as destructive for the tumor and protective for normal tissue simultaneously [51]. In this study, the principle of A. vulgaris extract duality was illustrated by the genoprotection of primary lymphocytes exposed to a certain dose range of the extract, while the same extract exerts cytotoxic potential against transformed cells in doses 5 to 15 times higher than those applied in the micronucleus assay. Bearing in mind that compounds such as quinic acid with a homing potential for cancer tissue are present in A. vulgaris extract, one can expect the multiple beneficial effects of its application in vivo. After collection, aerial parts of Alchemilla vulgaris L. were appropriately air-dried in a well-ventilated room (in shadow at 4 • C) and milled. The amount of 100 g of plant sample was extracted (period of 2 h) in hot ethanol at 60 • C (1 L) three times. After the separation of crude material, collected extracts were combined and evaporated in a vacuum at 60 • C by using a rotary evaporator. The obtained semisolid extract without solvent was sealed and stored at 4 • C for further analysis.

Phytochemical Analyses
The content of selected phytochemicals was determined by application of the standard spectrophotometric methods, namely Folin-Ciocalteu (total phenolics, TPC, New South Wales, Australia), aluminum chloride (total flavonoids, TFC, Daly City, CA, USA) and Arnow's method (total dihydroxycinnamic acid derivatives, HCA, Nashville, TN, USA), and expressed as mg/g equivalents of gallic acid (GAE), quercetin (QE) and chlorogenic acid (CGAE) calculated on dry weight (DW) of the sample respectively. For deeper phytochemical characterization, UHPLC-HRMS was performed.

UHPLC-HRMS Analysis
UHPLC-HRMS analysis was performed using a Thermo Scientific Dionex Ultimate 3000 RSLC (Germering, Bavaria, Germany) consisting of 6-channel degasser SRD-3600, high-pressure gradient pump HPG-3400RS, autosampler WPS-3000TRS, and column compartment TCC-3000RS coupled to a Thermo Scientific Q Exactive Plus (Bremen, Germany) equipped with heated electrospray ionization (HESI-II). UHPLC separation was achieved on a reversed-phase Kromasil Eternity XT C18 column (Nouryon, Göteborg, Sweden) (2.1 × 100 mm, 1.8 µm) equipped with precolumn SecurityGuard ULTRA UHPLC EVO C18 (Phenomenex, Torrance, CA, USA) maintained at 40 • C. The binary mobile phase consisted of A: 0.1% formic acid in water and B: 0.1% formic acid in acetonitrile. The run time was 34.5 min. The following gradient was used: the mobile phase was held at 5% B for 1 min, gradually turned to 30% B over 24 min, increased gradually to 40% B over 5 min, increased gradually to 95% B over 2.5 min, and held at 95% B for 2 min. The system was then turned to the initial condition of 5% B and equilibrated over 4.5 min. The flow rate and the injection volume were set to 300 µL/min and 2 µL, respectively. The tune parameters of the mass spectrometer were as follows: spray voltage, 2.5 kV; sheath gas flow rate, 38 arbitrary units (a.u.); auxiliary gas flow rate, 12 a.u.; capillary temperature and probe heater temperature, 320 • C; and S-lens RF level, 50. The acquisition was performed in the full-scan MS and data-dependent MS2 modes. Full-scan spectra over the m/z range of 100 to 1500 were acquired in negative ionization mode at a resolution of 70,000. Other instrument parameters for full MS mode were set as follows: automatic gain control (AGC) target, 3 × 10 6 ; maximum injection time (IT), 80 ms. For the ddMS2 mode, the instrument parameters were as follows: resolution, 17,500; AGC target, 1 × 10 5 ; maximum IT, 50 ms; Top5; isolation window, 2.0 m/z; stepped normalized collision energy (NCE), 20, 40, and 60 eV. Data acquisition and processing were carried out with Xcalibur 4.0 software (Thermo Scientific, Inc. Waltham, MA, USA).

Total Phenolic Content (TPC)
The Folin-Ciocalteu (FC) method was used for TPC determination according to [52]. Results were expressed as milligrams of ferulic acid equivalents (FAEs) per gram of dry weight (DW).

Total Flavonoid Content (TFC)
The determination of TFC was evaluated using the spectrophotometric method as described in [53]. TFC was determined using a calibration curve with quercetin (Q) as a standard, and the results were expressed as milligrams of quercetin equivalents (QEs) per gram of DW.

Total Dihydroxycinnamic Acid Derivative Content (HCA)
Total HCA content was estimated using the method described in [54]. The total HCA content in the extract was determined from the calibration curve with chlorogenic acid (CGA) as a standard. Results were expressed as milligrams of CGA equivalents (CGAEs) per gram of DW.

DPPH (2,2 -Diphenyl-1-picrylhydrazyl Radical) (DPPH) Assay
The determination of the free radical scavenging activity of the extracts was performed according to the method described in [55]. The percentage inhibition of DPPH was calculated by using the following formula: A b -the absorbance of blank; A s -the absorbance of the sample extract.

Ferric Reducing Power (FRP) Assay
The antioxidant activity of the A. vulgaris extract was determined by FRP assay according to the method previously described in [56]. Ascorbic acid (AA) was used as standard, and obtained results were expressed as milligrams of ascorbic acid equivalents (AAEs) per gram of DW.

Cupric Reducing Antioxidant Activity (CUPRAC) Assay
The CUPRAC assay was performed according to the procedure described in [57]. A calibration curve was prepared using different concentrations of ascorbic acid as a standard, and results were expressed as milligrams of ascorbic acid equivalents (AAEs) per gram of DW.

Total Antioxidant Capacity (TAC) Assay
The TAC assay was conducted using the method given in [58]. The antioxidant capacity was calculated according to a calibration curve prepared with ascorbic acid as standard. The results were expressed as milligrams of ascorbic acid equivalents (AAEs) per gram of DW.
In each method, all samples were analyzed in triplicates (n = 3). The absorbance of the resulting solution was measured with a UV/visible spectrophotometer. All cells were routinely maintained in HEPES-buffered RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 0.01% sodium pyruvate, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were grown at 37 • C in a humidified atmosphere with 5% CO 2 . The density of MCF-7, HCT116, and A375 at seeding time in 96-well plates for determination of cell viability was 4 × 10 3 cells/well, and that of A549 was 2 × 10 3 cells/well. For flow cytometric analyses concerning the A549 cell line in 6-well plates, the density was 7 × 10 4 cells/well.
Peritoneal exudate cells were collected by lavage with ice-cold PBS from the peritoneal cavity of C57BL/6 mice. Mice originated from our own animal facility at the Institute for Biological Research "Siniša Stanković" (IBISS)-National Institute of the Republic of Serbia, University of Belgrade (Belgrade, Serbia). Exudate cells were cultivated in HEPES-buffered RPMI-1640 medium supplemented with 5% heat-inactivated FBS, 2 mM L-glutamine, 0.01% sodium pyruvate, penicillin (100 units/mL), and streptomycin (100 µg/mL) at 37 • C in a humidified atmosphere with 5% CO 2 . Afterward, cells were counted, seeded in 96-well plates at a density of 1.5 × 10 5 cells/well, and left for 2 h to adhere. Prior to treatment, non-adherent cells were removed. The handling of animals and the study protocol were in agreement with the local guidelines and the European Community guidelines (EEC Directive of 1986; 86/609/EEC) and approved by the local Institutional Animal Care and Use Committee (IACUC). The approval for the experimental protocols (permission No. 323-07-120098/2020-05) was granted from the national licensing committee at the Department of Animal Welfare, Veterinary Directorate, Ministry of Agriculture, Forestry and Water Management of the Republic of Serbia.
Alchemilla vulgaris ethanolic extract stock solution was prepared in DMSO at a concentration of 200 mg/mL before the usage, and the final concentration of DMSO in working solutions was 1%.

Determination of Cell Viability by SRB and MTT Assays
All cell lines were seeded overnight and exposed to a wide range of concentrations of A. vulgaris extract for 72 h; after the incubation period, cell viability was assessed using SRB and MTT assays.
For detection of mitochondrial dehydrogenase activity, cells were incubated with MTT solution (0.5 mg/mL) for approximately half an hour until purple formazan crystals were formed. Afterward, the dye was discarded and DMSO was added to dissolve formazan. For the SRB assay, cells were fixed with 10% TCA for 2 h at 4 • C and stained with 0.4% SRB solution for 30 min at RT. Stained cells were dissolved in 1% acetic acid, washed, and dried overnight. The absorbance of dissolved dye (in 10 mM TRIS buffer for 20 min) was measured at 540 nm. Cell viability was calculated as a percentage of control that was arbitrarily set to 100%.

AnnV/PI, Apostat, and AO Staining
For all flow cytometric analyses, A549 cells were seeded overnight and treated with an IC50 dose of A. vulgaris extract (35 µg/mL) for 72 h.
For caspase activation detection, cells were stained with pan-caspase inhibitor Apostat in accordance with the manufacturer's protocol. For the detection of autophagosomes, cells were stained with a solution of 1 µg/mL AO for 15 min at 37 • C. For the detection of apoptotic cell death, cells were stained with 15 µg/mL Annexin V-FITC and 15 µg/mL PI and analyzed using CyFlow Space Partec using the PartecFloMax software (Munster, Germany).

CFSE Staining
Prior to treatment, A549 cells were stained with CFSE to a final concentration of 1 µM and incubated for 10 min at 37 • C. Afterward, cells were washed, seeded overnight, and treated with an IC 50 dose of A. vulgaris extract (35 µg/mL) for 72 h and then analyzed using flow cytometry.

Measurement of ROS/RNS Generation
By measuring the intensity of green fluorescence emitted by redox-sensitive dye DHR, the production of reactive oxygen and nitrogen species was detected. A549 cells were incubated with DHR for 20 min at 37 • C, seeded overnight, and treated with an IC 50 dose of A. vulgaris extract (35 µg/mL) for 72 h. At the end of the incubation period, cells were analyzed by flow cytometry.

DAPI Staining on Chamber Slides
To evaluate morphological signs of apoptosis, A549 cells were seeded in 4-chamber slides at 1 × 10 4 cells/well density and treated with an IC 50 dose (35 µg/mL) of A. vulgaris extract for 72 h. Afterward, cells were fixed with 4% (w/v) paraformaldehyde for 15 min at RT, washed, and covered with DAPI fluoromounth-G before analysis. Chamber slides were analyzed using Zeiss AxioObserver Z1 inverted fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany) at 400× magnification.

Cytokinesis-Blocked Micronucleus (CBMN) Assay
Venous blood samples were obtained with heparinized sterile vacutainers from 4 healthy female volunteers (2 × 5 mL from each) who had not been exposed to chemicals, drugs, or other substances. The volunteers signed informed consent and gave permission for the use of their blood for experimental purposes. The study complied with the code of ethics of the World Medical Association (Helsinki Declaration of 1975, as revised in 2013). The blood samples were obtained at the medical unit of the Nuclear Facilities of Serbia, the Institute of Nuclear Sciences "Vinca", in accordance with current (2005) Serbian health and ethical regulations.
For the preparation of MNs, the modified cytokinesis block method [59,60] was used. Cytochalasin B (Invitrogen-Gibco-BRL) was added to samples after 44 h of culture at a final concentration of 6 µg/mL, and the lymphocyte cultures were incubated for another 24 h. After 72 h, cells were washed with 0.9% NaCl (Merck, Sharp, & Dohme GMBH, Wien, Austria), collected by centrifugation, and treated with the hypotonic solution at 37 • C. The hypotonic solution consisted of 0.56% KCl + 0.9% NaCl (mixed in equal volumes). The cell suspension was prefixed in methanol/acetic acid (3:1), washed three times with fixative, and dropped onto a clean slide [59]. The slides were air-dried and stained with alkaline Giemsa (Sigma-Aldrich, St. Louis, MO, USA) (2%). At least 1000 binucleated (BN) cells per sample were scored, and MN was registered according to the criteria of [59,60].
Since micronucleus expression is dependent on cell proliferation, quantification of cell proliferation and cell death should be carried out to obtain a sound evaluation of cell kinetics and micronucleus frequencies. The CBPI was calculated as suggested in [59].
The number of binucleated cells with one, two, three, or more MNs were then tabulated. The data for each treatment were expressed as the frequency of MNs per 1000 binucleated cells.

Statistical Analysis
Student's t-test was used to evaluate the significance of differences between groups, and p-values of less than 0.05 were considered to indicate statistical significance.
For the micronucleus assay, the statistical significance of the difference between the data pairs was evaluated by analysis of variance (one-way ANOVA) followed by the Tukey test. Statistical difference was considered significant at p < 0.01. The calculated index is presented as the percentage of change between different groups.

Conclusions
The results of this study strongly support the historically collected data about the healing potential of Alchemilla vulgaris L. from Southeast Europe, which was traditionally used as a medicinal plant for centuries. This study confirmed that the ethanolic extract of Alchemilla vulgaris L. represents a valuable source of bioactive compounds with multiple beneficial biological properties, including strong antitumor activity and remarkable genoprotective features resulting, at least partly, from the strong antioxidant potential of this plant. Further research on the antitumor activity of lady's mantle should target the effects of individual components of its extract, as well as the effects of possible synergistic activity of different bioactive compounds, in addition to revealing their complex mechanisms responsible for anticancer action. All of the findings mentioned above make this plant a valuable candidate for further research in the field of drug discovery. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Data supporting obtained results can be obtained from the authors upon request.