Phytochemical Profiling of Extracts from Rare Potentilla Species and Evaluation of Their Anticancer Potential

Despite the common use of Potentilla L. species (Rosaceae) as herbal medicines, a number of species still remain unexplored. Thus, the present study is a continuation of a study evaluating the phytochemical and biological profiles of aqueous acetone extracts from selected Potentilla species. Altogether, 10 aqueous acetone extracts were obtained from the aerial parts of P. aurea (PAU7), P. erecta (PER7), P. hyparctica (PHY7), P. megalantha (PME7), P. nepalensis (PNE7), P. pensylvanica (PPE7), P. pulcherrima (PPU7), P. rigoi (PRI7), and P. thuringiaca (PTH7), leaves of P. fruticosa (PFR7), as well as from the underground parts of P. alba (PAL7r) and P. erecta (PER7r). The phytochemical evaluation consisted of selected colourimetric methods, including total phenolic (TPC), tannin (TTC), proanthocyanidin (TPrC), phenolic acid (TPAC), and flavonoid (TFC) contents, as well as determination of the qualitative secondary metabolite composition by the employment of LC–HRMS (liquid chromatography–high-resolution mass spectrometry) analysis. The biological assessment included an evaluation of the cytotoxicity and antiproliferative properties of the extracts against human colon epithelial cell line CCD841 CoN and human colon adenocarcinoma cell line LS180. The highest TPC, TTC, and TPAC were found in PER7r (326.28 and 269.79 mg gallic acid equivalents (GAE)/g extract and 263.54 mg caffeic acid equivalents (CAE)/g extract, respectively). The highest TPrC was found in PAL7r (72.63 mg catechin equivalents (CE)/g extract), and the highest TFC was found in PHY7 (113.29 mg rutin equivalents (RE)/g extract). The LC–HRMS analysis showed the presence of a total of 198 compounds, including agrimoniin, pedunculagin, astragalin, ellagic acid, and tiliroside. An examination of the anticancer properties revealed the highest decrease in colon cancer cell viability in response to PAL7r (IC50 = 82 µg/mL), while the strongest antiproliferative effect was observed in LS180 treated with PFR7 (IC50 = 50 µg/mL) and PAL7r (IC50 = 52 µg/mL). An LDH (lactate dehydrogenase) assay revealed that most of the extracts were not cytotoxic against colon epithelial cells. At the same time, the tested extracts for the whole range of concentrations damaged the membranes of colon cancer cells. The highest cytotoxicity was observed for PAL7r, which in concentrations from 25 to 250 µg/mL increased LDH levels by 145.7% and 479.0%, respectively. The previously and currently obtained results indicated that some aqueous acetone extracts from Potentilla species have anticancer potential and thus encourage further studies in order to develop a new efficient and safe therapeutic strategy for people who have been threatened by or suffered from colon cancer.


Introduction
Cancer, a non-infectious disease, is one of the most dreadful diagnoses that severely impacts a patient's life quality. Unfortunately, cancer is a significant and increasing cause

Examination of the Anticancer Potential of Extracts
In the first step, the extract's influence on both human colon epithelial cell line CCD841 CoN as well as human colon adenocarcinoma cell line LS180 was examined using an MTT assay. Studies were conducted after 48 h of the cells being exposed to either a culture medium (control) or extracts (25-250 µg/mL). As presented in Figure 1 and Table 3, all the investigated extracts inhibited the metabolic activity of both normal and cancer cells, and the observed effect was dose-dependent. The most significant anticancer effect was presented by extracts PAL7r and PFR7, which, at the highest tested concentration, deceased LS180 cells' proliferation by 91.3% (IC 50 PAL7r LS180 = 82 µg/mL) and 94.8% (IC 50 PFR7 LS180 = 89 µg/mL), respectively. On the contrary, the weakest influence on the metabolic activity of colon cancer cells was noted after treatment with PTH7 and PRI7, which, at a concentration of 250 µg/mL, inhibited cell viability by 58.7% (IC 50 PTH7 LS180 = 225 µg/mL) and 57.9% (IC 50 PRI7 LS180 = 213 µg/mL), respectively. The strongest reduction (by 36.7%) of the viability of colon epithelial cells was caused by both PME7 and PHY7 (IC 50 PME7 CCD841 CoN = 380 µg/mL; IC 50 PHY7 CCD841 CoN = 489 µg/mL), while the weakest effect, as reflected by the IC 50 value, was shown by PRI7 (IC 50 PRI7 CCD841 CoN = 2402 µg/mL).
Used as a positive control for the experiment, 5-fluorouracil (5-FU) at a concentration of 25 µM decreased the metabolic activity of CCD841 CoN and LS180 by 22.2% and 46.2%, respectively. All the investigated extracts at the highest tested concentrations revealed a stronger anticancer effect than 5-FU. Seven of twelve extracts inhibited LS180 cells' viability better than 5-FU, when used at lower concentrations; the beneficial effect of PER7r, PHY7, PME7, and PPE7 was observed at concentrations of 150 and 250 µg/mL, while the beneficial effect of PAL7r, PFR7, and PNE7 was observed at concentrations from 100 to 250 µg/mL. In the case of CCD841 CoN cells, only 3 out of 12 of the investigated extracts inhibited the metabolic activity of colon epithelial cells stronger than 25 µM 5-FU: PAL7r (250 µg/mL); PHY7 (250 µg/mL); PME7 (150 and 250 µg/mL).
The obtained results for the MTT assay may be strongly associated with high TPrC, especially in the PAL7r, PER7r, and FFR7 extracts. On several occasions, proanthocyanidins were reported to have a strong influence on colon cancer cell viability. Especially oligomeric proanthocyanidins from grape seeds (Vitis vinifera L., Vitaceae), which induce the apoptotic cell death of Caco-2 (human colorectal adenocarcinoma) cells manifested by nuclear condensation, caspase-3 and PARP cleavage, and formation of apoptotic bodies [31]. Additionally, proanthocyanidins from hops (Humulus lupulus L., Cannabaceae) increased the intracellular formation of reactive oxidative species, which was manifested by the augmented accumulation of protein carbonyls and induced cytoskeletal disorganisation of human colon cancer cell line HT-29 [32]. However, in a comparison with a previous study, all extracts exerted a weaker effect on cancer cell viability than extracts obtained from five out of six tested aqueous acetone extracts, namely, P. argentea, P. grandiflora, P. norvegica, P. recta, and P. rupestris [10]. This difference may be associated with the lower TPC and TTC obtained herein. Ellagitannins display great chemopreventive and chemotherapeutic activities. Among them, agrimoniin, the main ellagitannin present in all extracts, except PAL7r, was shown to have prominent anticancer, antioxidant, and anti-inflammatory activities [33]. It is widely recognised that there is a strict correlation between chronic inflammation and colorectal cancerogenesis [34]. Preclinical and clinical studies showed that non-steroidal and anti-inflammatory drugs are effective in preventing the formation of colorectal tumours; however, there are limitations due to severe and fatal side effects, such as gastric bleeding, ulcers, and renal toxicity [35]. Phytochemicals have fewer side effects compared with synthetic drugs, which is advantageous. A study conducted by Shi and co-authors revealed that the use of lyophilised strawberries (Fragaria x ananasa L., Rosaceae) containing agrimoniin as the second-most-abundant phytochemical, in an inflammationinduced colorectal carcinogenesis model, led to downregulating the mRNA expression of the proinflammatory mediators, such as COX-2, iNOS, IL-1β, IL-6, and TNF-α [36]. Moreover, in two consecutive studies, an agrimoniin-enriched fraction from the underground parts of P. erecta showed the dose-dependent inhibition of UVB-induced or TNF-α stimulated IL-6 and PGE 2 production as well as reduced NFκB activation in HaCaT cells (human keratinocytes). Further, a UV erythema study in healthy volunteers revealed that an agrimoniin-enriched fraction significantly inhibited the UVB-induced inflammation process [37,38].
bioavailability of large ellagitannins is generally low. Therefore, the method of application is limited to topical application. The gut microbiota metabolise ellagitannins and ellagic acid to produce a series of bioavailable metabolites, known as urolithins. Urolithins possess a series of biological activities, such as anti-inflammatory, antioxidant, anticancer, and immunomodulatory activities. The chemopreventive effects of urolithins were extensively studied in several models, including prostate and colorectal cancer models. Urolithins were shown to inhibit colon cancer cell growth in a dose-dependent manner, alter the expression of the genes and proteins modulating the cell cycle, and induce apoptosis [46]. Notably, a clinical study on the aerial parts of P. anserina and the rhizomes of P. erecta confirmed the formation of urolithins in ex vivo conditions [47]. Figure 1. Influence of obtained extracts on the viability of CCD841 CoN and LS180 cell lines. The cells were exposed for 48 h to the culture medium alone (control). extract at concentrations of 25-250 µg/mL. or 25 µM 5-fluorouracil (5-FU; positive control). Metabolic activity of investigated cell lines in response to tested compounds was examined photometrically by MTT assay. Results are presented as mean ± SEM of at least 5 measurements. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control. # p < 0.05; ## p < 0.01; ### p < 0.001 vs. positive control. ^^ p < 0.01; ^^^ p < 0.001 colon cancer cells treated with extract/5-FU vs. colon epithelial cells exposed to the extract/5-FU at the corresponded concentration; one-way ANOVA test; post-test: Tukey's. Figure 1. Influence of obtained extracts on the viability of CCD841 CoN and LS180 cell lines. The cells were exposed for 48 h to the culture medium alone (control). extract at concentrations of 25-250 µg/mL. or 25 µM 5-fluorouracil (5-FU; positive control). Metabolic activity of investigated cell lines in response to tested compounds was examined photometrically by MTT assay. Results are presented as mean ± SEM of at least 5 measurements. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control. # p < 0.05; ## p < 0.01; ### p < 0.001 vs. positive control.ˆˆp < 0.01;ˆˆˆp < 0.001 colon cancer cells treated with extract/5-FU vs. colon epithelial cells exposed to the extract/5-FU at the corresponded concentration; one-way ANOVA test; post-test: Tukey's. In the next step, the antiproliferative activity of Potentilla L. extracts was examined in both the normal and cancer cell lines using a BrdU assay (Figure 2 and Table 1). All the investigated extracts decreased DNA synthesis in the colon cancer cells in a dosedependent manner. Nevertheless, a significant decrease in LS180 cells' proliferation in response to the extract, for the whole range of tested concentrations, was only observed for PAL7r, PFR7, and PER7r, which, at concentrations of 100, 150, and 250 µg/mL, reduced DNA synthesis by around 80%. Furthermore, the aforementioned extracts were characterised by the lowest IC 50 values (IC 50 PAL7r LS180 = 52 µg/mL; IC 50 PFR7 LS180 = 50 µg/mL; IC 50 PER7r LS180 = 54 µg/mL). On the contrary, the lowest antiproliferative abilities were revealed by PAU7, which, even at the highest tested concentration, decreased DNA synthesis in LS180 cells by only 14.9% (IC 50 PAU7 LS180 = 1495 µg/mL). The antiproliferative effect of the examined extracts was also observed in colon epithelial cells; however, the observed effect was weaker than in cancer cells. The only extract that did not affect divisions of CCD841 CoN was PER7, which was characterised by the highest IC 50 value of 3705 µg/mL. On the contrary, the most significant changes in normal cells were observed in response to PAL7r, PFR7, and PER7r, which, at the highest tested concentration, reduced the proliferation of epithelial cells by 36.1%, 38.3%, and 43.9%, respectively (IC 50 PAL7r CCD841 CoN = 412 µg/mL; IC 50 PER7 CCD841 CoN = 282µg/mL; IC 50 PER7r CCD841 CoN = 337 µg/mL). As presented in Figure 2, 25 µM 5-fluorouracil (5-FU) decreased DNA synthesis in the investigated cell lines to 90.7% (CCD841 CoN) and 29.7% (LS180). The antiproliferative effect of 5-FU recorded in colon cancer cells was significantly stronger than the changes induced by most of the examined extracts (9 of 12); however, PAL7r, PFR7, and PER7r, in concentrations from 100 to 250 µg/mL, decreased DNA synthesis more than 5-FU. On the contrary, data collected from colon epithelial cells revealed that most of the investigated extracts (PAL7r, PER7r, PFR7, PHY7, PME7, PNE7, PPE7, PPU7, PRI7, and PTH7) at higher concentrations inhibited DNA synthesis stronger than 25 µM 5-FU, while the antiproliferative effect of PFR7 for the whole range of tested concentrations was higher than the changes induced by analysed cytostatic. However, the presented results correspond with data from our previous study, showing that tested Potentilla species possess similar anticancer potential; moreover, for the PAL7r, PER7r, and PFR7 extracts, the results from a BrdU assay were significantly higher than those for all other tested samples [10]. The observed effect may be attributed to the high TPrC values. Kresty and co-authors found that a cranberry (Vaccinium macrocarpon Aiton, Ericaceae) proanthocyanidin-rich fraction significantly inhibited the viability and proliferation of human oesophageal adenocarcinoma SEG-1 cells. The mechanism involved cell cycle arrest in the G1 phase as well as a significant apoptosis induction [39]. Notably, the antiproliferative effect of other extracts may be connected with the presence of ellagitannins and the main product of their decomposition, namely, ellagic acid. The anticancer mechanism of ellagic acid is multidirectional. A study conducted on human colorectal carcinoma HCT-116 and the Caco-2 cell line revealed that ellagic acid induced cell cycle arrest in the G phase, reduced proliferating cell nuclear antigen (PCNA) expression and mitotic activity, and induced apoptosis via increasing the expression of caspase-8 and Bax [40]. Additionally, a further study conducted on HCT-116 cells revealed the involvement of ellagic acid in the decreased gene expression of signalling pathways' proteins such as mitogen-activated protein (MAPK), p53, PI3K-Akt, and TGF-β [41]. Recently, Han and co-authors found that tiliroside acted as an inhibitor of carbonic anhydrase XII (CAXII), a membrane enzyme that produces a favourable intracellular pH and sustains optimum P-glycoprotein (Pgp) efflux activity in cancer cells. Moreover, tiliroside downregulated E2F1 and E2F3 expression and promoted caspase-3 activity [39]. In addition, the meta-analysis revealed that a high intake of flavonoids, such as quercetin and kaempferol derivatives, in the diet may decrease the risk of colon cancer [42]. Antiproliferative effect of extracts on CCD841 CoN and LS180 cell lines. The cells were exposed for 48 h to the culture medium alone (control). extract at concentrations of 25-250 µg/mL. or 25 µM 5-fluorouracil (5-FU; positive control). The antiproliferative impact of investigated compounds was measured using BrdU assay (incorporation of BrdU to newly synthesised DNA). Results are presented as mean ± SEM of at least 4 measurements. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control. # p < 0.05; ## p < 0.01; ### p < 0.001 vs. positive control. ^ p < 0.05; ^^ p < 0.01; ^^^ p < 0.001 colon cancer cells treated with extract/5-FU vs. colon epithelial cells exposed to the extract/5-FU at the corresponded concentration; one-way ANOVA test; post-test: Tukey's.
Extract cytotoxicity was also examined in both normal and cancer colon cell lines using an LDH (lactate dehydrogenase)-based assay (Figure 3). Most of the examined extracts were not cytotoxic against human colon epithelial cells; however, PME7 was, for the whole range of tested concentrations, while PAL7r, PER7, and PHY7, in concentrations from 100 to 250 µg/mL, damaged the membranes of epithelial cells. The indicated extracts at the highest tested concentration increased the LDH level by an average of 11%. Studies conducted on colon cancer cells showed the cytotoxic effect of all the examined extracts for the whole range of tested concentrations. The strongest damage of cancer cell membranes was caused by PAL7r, which in concentrations from 25 to 250 µg/mL increased the LDH level by 145.7% and 479.0% respectively. The weakest cytotoxic effect was noted in colon cancer cells treated with PRI7, PPU7, and PTH7, which, at the highest tested concentration, caused an increase in the LDH level by 245.1%, 254.7%, and 256.0%, respectively. An LDH assay showed that 5-FU in a concentration of 25 µM was not cytotoxic against colon epithelial, while LDH releases were increased in colon cancer cells by 13.4%. All the investigated extracts damaged the colon cancer cell membranes significantly more than 5-FU. For CCD841 CoN cells, significant differences in the LDH concentration between 25 µM 5-FU and the examined extracts were observed in the case of four extracts (PME7, PAL7r, PER7, and PHY7), for which the cytotoxic impact on colon epithelial cells was reported above. The results of the LDH assay are presumably directly associated with the TTC in the investigated samples. Tannins, due to their specific chemical structure, are known to affect the physical properties of membranes, initiate membrane protein aggregation, increase bilayer adhesion, and regulate cell metabolism [43,44]. The most abundant hydrolysable tannin present in most extracts, agrimoniin, induces the intrinsic pathway of apoptosis, directly influencing the permeability of the mitochondrial membrane via the activation of the mitochondrial permeability transition pore (MPTP) [45]. However, further in vivo studies are required to evaluate the exact mechanism of action. The bioavailability of large ellagitannins is generally low. Therefore, the method of application is limited to topical application. The gut microbiota metabolise ellagitannins and ellagic acid to produce a series of bioavailable metabolites, known as urolithins. Urolithins possess a series of biological activities, such as anti-inflammatory, antioxidant, anticancer, and immunomodulatory activities. The chemopreventive effects of urolithins were extensively studied in several models, including prostate and colorectal cancer models. Urolithins were shown to inhibit colon cancer cell growth in a dose-dependent manner, alter the expression of the genes and proteins modulating the cell cycle, and induce apoptosis [46]. Notably, a clinical study on the aerial parts of P. anserina and the rhizomes of P. erecta confirmed the formation of urolithins in ex vivo conditions [47].  The cells were exposed for 48 h to the culture medium alone (control). extract at concentrations of 25-250 µg/mL. or 25 µM 5-fluorouracil (5-FU; positive control). Extracts' cytotoxicity (level of LDH released into the cell culture medium from damaged cell membranes) was measured using an LDH assay. Results are presented as mean ± SEM of at least 3 measurements. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. control. # p < 0.05; ## p < 0.01; ### p < 0.001 vs. positive control. ^^^ p < 0.001 colon cancer cells treated with extract/5-FU vs. colon epithelial cells exposed to the extract/5-FU at the corresponded concentration; one-way ANOVA test; post-test: Tukey's.

Plant Materials and Extraction Procedure
Plants used to obtain material for investigations come from the Medicinal Plant Garden at the Medical University of Białystok (Białystok, Poland) and were collected in June-August 2017-2020. Plants were carefully identified by one of the authors (M.T.), and individual voucher specimens were deposed at the Herbarium of the Department of Pharmacognosy, Medical University of Białystok (Białystok, Poland). Plant material was dried at room temperature in the shade and air temperature and subsequently finely ground with an electric grinder. Accurately weighed 2 g of each powdered dry plant material were separately extracted using an ultrasonic bath (Sonic-5, Polsonic, Warszawa, Poland) with 70% acetone at a controlled temperature (40 ± 2 • C) for 45 min in a 1:75 (w:v) solvent ratio to obtain raw extracts. Subsequently. extracts were evaporated to dryness, diluted with water (50 mL). and successively portioned between chloroform (10 × 20 mL). Afterwards. purified extracts were freeze-dried. The list of obtained aqueous acetone extracts from selected Potentilla species detailing plant species, voucher specimen, the parts used and extraction yields are presented in Table 4.

Determination of Total Phenolic (TPC) and Total Tannin Content (TTC)
The content of total phenolic compounds was measured using standard the Folin-Ciocalteu colourimetric method, with slight modification according to [29]. The content total tannin determination was carried out using the hide powder-binding method and Folin-Ciocalteu assay reported in the corresponding monograph in the European Pharmacopoeia 10th ed. [52]. The absorbance was measured at 760 nm using a EPOCH2 BioTech (Winooski, VT, USA) microplate reader. The obtained results for were expressed as milligrams of gallic acid equivalents per gram of extract (mg GAE/g extract). The determination was repeated at least in triplicate for each sample solution.

Determination of Total Proanthocyanidin Content (TPrC)
Total proanthocyanidin content was analysed using the procedure based on the previously published protocol [53]. The analysis was carried out by mixing 50 µL of the sample solution (1 mg/mL) dissolved in methanol and 250 µL of 0.1% methanolic solution of 4-dimethylamino-cinnamaldehyde (DMCA) reagent in 6M HCl. After incubation of the mixture at room temperature for 15 min, the absorbance was measured at 635 nm, and results were expressed as milligrams of catechin equivalents per gram of extract (mg CE/g extract). The determination was repeated at least five times for each sample solution.

Determination of Total Phenolic Acid Content (TPAC)
Total phenolic acid content was estimated with the procedure using Arnov's reagent (1 g of sodium molybdate and 1 g of sodium nitrate dissolved in 10 mL of distilled water) [54]. Each time the tested solution (30 µL) was mixed with 180 µL of water, 30 µL of 0.5 M HCl, 30 µL of Arnov's reagent, and 30 µL of 1 M NaOH were sequentially added to the microplate well, and then it was incubated for 10 min at ambient temperature. Afterwards, the absorbance was measured at 490 nm, and results were expressed as milligrams of caffeic acid equivalents per gram of extract (mg CAE/g extract). The determination was repeated at least three times for each sample solution.

Determination of Total Flavonoid Content (TFC)
Total flavonoid content was estimated by the previously described colourimetric method [29]. Each aliquot (100 µL) was mixed with aluminum chloride (AlCl 3 ) solution (100 µL, 2% w:v). After incubation of the mixture at room temperature for 10 min, the absorbance was measured at 415 nm, and results were expressed as milligrams of rutin equivalents per gram of extract (mg RE/g extract). The determination was repeated at least three times for each sample solution.

LC-HRMS Profiling of Extracts
The separation and qualitative evaluation of each extract were conducted using a Kinetex XB-C18 column (150 × 2.1 mm, 1.7 µm, Phenomenex, Torrance, CA, USA) and Agilent 1260 Infinity LC chromatography system coupled to a photo-diode array (PDA) detector and 6230 time-of-flight (TOF) mass spectrometer (Santa Clara, CA, USA). A detailed description of the execution of the above-mentioned assay was presented in the previous study [10].

Cell Cultures
For the cell culture study, human colon adenocarcinoma cell line LS180 and human colonic epithelial cell line CCD841 CoN were purchased from the European Collection of Cell Cultures (ECACC, Centre for Applied Microbiology and Research, Salisbury, UK) and American Type Culture Collection (ATCC, Menassas, VA, USA), respectively. LS180 cells and CCD841 CoN cells were maintained in Dulbecco s Modified Eagle s Medium/Nutrient Mixture F-12 Ham and Dulbecco's Modified Eagle's Medium (DMEM), respectively. Then, 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 g/mL) were added to the cell culture media. Cells were incubated in a humidified atmosphere of 95% air and 5% CO 2 at 37 • C.

Evaluation of the Anticancer Potential of Extracts
Examination of the anticancer potential of extracts was conducted simultaneously on both cancer (LS180) and normal (CCD841 CoN) colon cells. Cells at a density of 5 × 104 cells/mL were plated on 96-well plates. The next day, the cell growth medium was exchanged for fresh medium supplemented with investigated extracts or 25 µM 5fluorouracil (5-FU). After 48 h of treatment, compound impacts on cell membrane integrity, metabolic activity, and DNA syntexis were examined using LDH, MTT, and BrdU assays, respectively. The description of the execution of indicated tests was previously presented [10].

Statistical Analysis
The data were presented as the mean ± standard error of mean (SEM). Statistical analyses were carried out using one-way ANOVA with Tukey's post hoc test and column statistics. Significance was accepted at p < 0.05. The IC 50 value (concentration causing proliferation inhibition by 50% compared to control) was calculated according to the method of Litchfield and Wilcoxon [55] using GraphPad Prism 5.

Conclusions
In conclusion, the presented study reports, for the first time, an analysis of the LC-HRMS profile of aqueous acetone extracts from rare Potentilla species. The analysis revealed a series of marker metabolites such as agrimoniin, pedunculagin, dimeric and trimeric Btype procyanidins, tiliroside, astragalin (kaempferol 3-O-glucoside), hyperoside (quercetin 3-O-galactoside, ellagic acid, and tri-coumaroyl spermidine. The performed studies revealed that all of the investigated acetone extracts obtained from rare Potentilla species decreased the viability and proliferation of human colon adenocarcinoma LS180 cells. Nevertheless, most of the investigated extracts also decreased metabolic activity and DNA synthesis in human colon epithelial CCD841 CoN cells, and 4 out of 12 of the tested extracts (PAL7r, PER7, PHY7, and PME7) showed cytotoxic effects against normal epithelial cells. Despite the fact that the investigated extracts affected both normal and cancer colon cells, the LS180 cells were more sensitive to tested extracts. Considering the data obtained from all the performed studies, the 2 of the 12 investigated extracts (PFR7 and PER7r) revealed the greatest chemopreventive potential, as manifested by the effective elimination of colon cancer cells, which caused both damage to their cell membranes and inhibition of their proliferation and metabolic activity, with a simultaneous lack of any cytotoxic effect on normal colon epithelial cells and a significantly weaker effect on their metabolism and DNA synthesis compared to cancer cells. The previous [10] and currently obtained results indicated that some acetone extracts from Potentilla species have anticancer potential, however, additional animal and clinical studies, especially including the influence of intestinal flora are required to verify discovered beneficial properties of investigated extracts. Nevertheless, discovered selectivity of the anticancer effects of tested extracts encourages further studies to develop a new efficient and safe therapeutic strategy for people who have been threatened by or suffered from colon cancer.

Data Availability Statement:
The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.