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

Abstract

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.

1. 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 of death worldwide. The European Cancer Information System (ECIS) estimated an increase in new cases of cancer in the European Union (EU-27) from 2.68 million in 2020 to 3.24 million in 2040, a 21% increase, while the cancer-related death toll is estimated to increase from 1.26 million to 1.66 million cases, a 31.8% increase. Colorectum cancer is the second-most-diagnosed cancer type in EU-27 countries, with over 0.34 million cases in 2020; however, in 2040, it will overtake breast cancer as the most commonly diagnosed cancer type with over 0.43 million cases [1]. The most frequently used method to treat early-stage colorectal cancer is surgical resection, which effectively relieves the patient’s symptoms. However, approximately 25% to 30% of patients after successful surgery will develop metastases within 5 years [2]. Moreover, in the further stages, unresectable metastatic cancer systemic therapy includes chemotherapy, radiotherapy, immunotherapy, and biological therapy, such as antibodies to cellular growth factors, as well as their combinations [3]. Unfortunately, these treatment methods are inextricably linked with many side effects, such as pain, emotional stress, fatigue, a negative impact on fertility, and subsequent cancers [4]. Biologically active molecules in medicinal plants can be employed to reduce side effects and support the efficacy of the therapy. Notably, Potentilla species are widely used in traditional medicine for the treatment of dysentery, diarrhoea, diabetes mellitus, unspecified forms of cancer, and inflammation of the skin [5,6]. The pharmacological properties of Potentilla species stem from their secondary metabolite composition, which includes a predominant presence of polyphenols, such as hydrolysable and condensed tannins, flavonoids, and phenolic acid, as well as triterpenoids. These substances are associated with antioxidant, anti-inflammatory, and antimicrobial properties [5]. Numerous in vitro experiments on compounds obtained from Potentilla species have shown efficacy against various cancer cell lines, e.g., methanol extract from P. discolor inhibited the proliferation and induced the apoptosis of MC3 and YD-15 (human mucoepidermoid carcinoma) [7], ethyl acetate extracts from P. recta and P. astracanica decreased viability of HEp-2 (human cervix carcinoma) [8], and selected extracts and fractions from aboveground materials of P. alba significantly reduced the viability and proliferation of HT-29 (human colon adenocarcinoma) [9]. In a previous study, we demonstrated that aqueous acetone extracts from the aerial parts of selected Potentilla species showed great chemopreventive potential by decreasing the viability and proliferation of LS180 (human colon adenocarcinoma) cells, simultaneously causing substantial damage to their cell membranes while having a significantly weaker impact on normal colon epithelial cell line CCD841 CoN [10]. The present study is a continuation of that previous investigation conducted by the authors, concerning an assessment of the cytotoxicity and antiproliferative effect of aqueous acetone extracts from selected, rare Potentilla species against human colon cancer cell line LS180 and normal colon epithelial cell line CCD841 CoN. Additionally, identification of the marker metabolites present in extracts using LC–HRMS analysis was conducted to reveal and validate correlations between the qualitative chemical composition of the investigated samples and possible mechanisms of action.

2. Results and Discussion

2.1. Determination of Total Secondary Metabolites Content

Polyphenols are among the major secondary metabolites that are accountable for the pharmacological activities of plant-based preparations. The major group of polyphenols include flavonoids, phenolic acids, hydrolysable and condensed tannins, lignans, and stilbenes [11]. Potentilla species are well-known for their abundance of tannins and flavonoids, which contribute to certain traditional applications aimed at tackling diarrhoea, microbial infections, inflammations of the upper and lower gastrointestinal tract, diabetes mellitus, etc. [5,12]. In our study, extracts from the aerial and underground parts of common and rare Potentilla species were prepared using 70% acetone and were quantitative assessed for the general polyphenolic classes contents using colourimetric methods. The level of phenolic compounds in the extracts from selected Potentilla species are presented in

Table 1. Total phenolic (TPC), tannin (TTC), proanthocyanidin (TPrC), phenolic acid (TPAC), and flavonoid contents (TFC) of aqueous acetone extracts.

Samples	TPC (mg GAE/g Extract) 1	TTC (mg GAE/g Extract) 1	TPrC (mg CE/g Extract) 2	TPAC (mg CAE/g Extract) 3	TFC (mg RE/g Extract) 4
PAL7r	268.6 ± 6.9	237.6 ± 5.7	72.6 ± 2.5	221.1 ± 7	15 ± 0.3
PAU7	148.4 ± 2.3	129.2 ± 2	3.4 ± 0.1	44.2 ± 1.4	59.7 ± 1.3
PER7	201.2 ± 4.3	169.2 ± 7	2.1 ± 0.1	59.9 ± 1.3	54.9 ± 0.4
PER7r	326.3 ± 3.5	269.8 ± 2.4	61.6 ± 1.1	263.5 ± 7.5	11 ± 0.1
PFR7	240.1 ± 6.1	178.7 ± 5.5	53.6 ± 0.9	197.8 ± 6.2	94.6 ± 2.4
PHY7	199.2 ± 1.7	178.2 ± 3.9	1.6 ± 0.1	44 ± 1.1	113.3 ± 1.5
PME7	195.3 ± 4.4	168.5 ± 3.6	13.1 ± 0.4	80.8 ± 2	84.6 ± 0.1
PNE7	188.8 ± 2.5	163.5 ± 0.5	1.1 ± 0.1	33.4 ± 0.3	66.5 ± 2.5
PPE7	218.9 ± 1.8	196 ± 3.1	0.2 ± 0.1	50.5 ± 0.5	108.2 ± 0.5
PPU7	151.5 ± 2.4	135.9 ± 2.4	5.5 ± 0.1	50.2 ± 2.1	64.9 ± 0.6
PRI7	212.2 ± 5.5	170.5 ± 4.4	5.6 ± 0.6	58.1 ± 1.7	84.4 ± 0.7
PTH7	149.8 ± 2.3	132.6 ± 2.3	4.9 ± 0.1	58.8 ± 2.5	76.4 ± 1.6

¹ GAE—gallic acid equivalent; ² CE—catechin equivalent; ³ CAE—caffeic acid equivalent; ⁴ RE—rutin equivalent. All values represent the mean ± standard deviation of three replicates for each sample (n = 3).

. Extracts from the underground parts, namely, PAL7r and PER7r, were found to contain the highest total phenolic (TPC) and total tannin (TTC) contents (268.63, 237.56, and 326.28, 269.79 mg gallic acid equivalent (GAE)/g extract, respectively). On the other hand, among extracts from the aerial parts, PFR7 and PPE7 had the highest TPC and TTC values (240.1, 178.65, and 218.85, 195.97 mg GAE/g extract, respectively), while PAU7 and PTH7 revealed the lowest TPC and TTC values (148.38, 129.2, and 149.77, 132.55 mg GAE/g extract, respectively). Moreover, PFR7 was found to contain the highest total proanthocyanidin content (TPrC) (53.59 mg catechin equivalent (CE)/g extract), notably higher than that of other herb extracts. According to our previous study and the results herein, extracts from rhizomes, namely, PAL7r and PER7r, had remarkably higher proanthocyanidin contents than their above-ground counterparts (72.63 and 61.61 vs. 21.28 and 2.05 mg CE/g extract, respectively) [12]. Moreover, PAL7r and PER7r had the highest total phenolic acid content (TPAC), followed by PFR7 (221.08, 263.54, and 197.83 mg caffeic acid equivalent (CAE)/g extract, respectively). On the contrary, PAL7r and PER7r had the lowest total flavonoid content (TFC) values, which were significantly lower than those of all other extracts. PHY7 and PPE7 revealed the highest TFC values (113.29 and 108.2 mg rutin equivalent (RE)/g extract, respectively). All the obtained results were significantly higher than the values available in the literature data reported for various extracts from the aerial parts of P. erecta, P. fruticosa, P. nepalensis, P. pensylvanica, and P. thuringiaca [13,14,15]. Notably, the selection of the solvent in the extraction process is a crucial factor in the explanation of those differences. An aqueous acetone solvent extracts much fewer non-phenol compounds, such as carbohydrates, than methanol and water, which results in higher TPC and TFC values [16]. Moreover, aqueous acetone was reported as an excellent solvent for extracting higher molecular weight flavonoids and proanthocyanidins [17]. The aforementioned solvent prevents the decomposition of hydrolysable tannins during the extraction process, leading to a higher tannin content in the obtained extracts [18].

2.2. LC–HRMS Qualitative Analysis of Selected Extracts

The identification of the secondary metabolite composition of the aqueous acetone extracts of selected Potentilla species using LC–HRMS (liquid chromatography–high-resolution mass spectrometry) analysis demonstrated the presence of 198 compounds. Among them, three groups of phenolic compounds were dominant in the analysed extracts: tannins, flavonoids, and phenolic acids. Monomeric and dimeric ellagitannins, such as agrimoniin, sanguinis and pedunculagin, are important chemophenetic markers in the Rosaceae family, especially in the Potentilla, Rubus, and Fragaria genera [19]. The chromatographic analysis reported herein led to the identification of a series of hydrolysable tannins that are represented by ellagitannin derivatives, such as laevigatin isomers (84, 109, 114, 124, and 128), laevigatin E isomers (37 and 40), agrimoniin (162) and its structural isomer (151), agrimonic acid A or B (102), galloyl-HHDP-glucose (16, 21, 43, and 48), digalloyl-HHDP-glucose (33 and 60) and trigalloyl-HHDP-glucose (131 and 133), galloyl-bis-HHDP-glucose (108, 118, and 144), ellagic acid (135) and its O-pentosides (132 and 163), O-hexosides (73, 97, and 101), and uronic acid (82, 95, and 130) derivatives. The analysis indicated that the one of the most abundant phytochemicals in all the extracts, except PAL7r, was agrimoniin. Agrimoniin has been frequently described as the major phenolic compound in several Potentilla species, such as P. argentea, P. anserina, P. grandiflora P. kleiniana P. norvegica, P. recta, and P. rupestris [10,20,21,22]. Other present ellagitannins, namely, leavigatins and agrimonic acid, are formed from the partial hydrolysis of agrimoniin (dehydrodigalloyl-di-(bis-HHDP-glucose)) [23]. Furthermore, few degradation products of hydrolysable tannins degradation, such as ellagic acid (135), brevifolincarboxylic acid (46) and its structural isomer (50), and brevifolin (83), were found. Gallotannins were present in a few extracts, which showed the presence of di-, tri-, tetra-, and pentagalloylglucose isomers (35, 36, 80, 86, 103, 137, and 168). However, the analysis revealed the absence of hydrolysable tannins in PAL7r. These findings are in agreement with the previous study, which demonstrated the absence of these metabolites in the aerial parts of P. alba [9]. Moreover, the analysis revealed the presence of condensed tannins, especially in PAL7r, such as catechin (28), epicatechin (61), and their glucosides (11, 22, 23, 41, and 106), as well as products of their polymerisation, such as A-type procyanidins (24, 54, 71, 90, 96, and 110) and dimeric (66, 88, and 107), trimeric (7, 42, 45, and 64) and tetrameric (56 and 93) B-procyanidins, including procyanidin B1 (25), procyanidin B2 (47), procyanidin B3 (27), procyanidin C1 (94), and procyanidin C2 (34).

Based on the chromatographic profiles, a number of flavonoids were detected and characterised, including apigenin (92, 119, 161, 166, 184, and 185) as well as isorhamnetin (87, 91, 98, 100, 125, 150, 158, 167, 169, 171–173, 179, 182, 187, 188, 191, 194, and 196), naringenin (180), kaempferol (62, 67, 72, 78, 81, 104, 113, 117, 140, 142, 143, 147, 148, 153, 155, 157, 159, 160, 164, 165, 170, 175, 176, 178, 190, 192, and 193), quercetin (39, 44, 51, 53, 55, 58, 59, 63, 74, 79, 85, 99, 105, 111, 116, 120–122, 126, 127, 129, 134, 136, 138, 139, 141, 145, 149, 152, 154, 156, 177, and 186), acacetin (183), and tricin (189 and 195) derivatives. From a chemophenetic perspective, a few of them may be useful as chemical markers of the Potentilla genus, such as both isomers of tiliroside (190), astragalin (kaempferol 3-O-glucoside) (155), isorhamnetin 3-O-glucoside (169), kaempferol 3-O-glucuronide (157), avicularin (quercetin 3-O-arabinoside) (149), hyperoside (quercetin 3-O-galactoside) (139), isoquercitrin (quercetin 3-O-glucoside) (136), and rutin (quercetin 3-O-rutinoside) (138), which were previously reported to be present in at least one of the Potentilla species investigated to date [5,6,10]. The analysis also revealed the presence of phenolic acids, such as gallic acid (1), caffeic acid (29) and its derivatives (5, 9, 10, 15, 22, 65, and 75), coumaric acid (12, 17, 57, and 197), dihydroxybenzoic acid (13), and syringic acid (89) derivatives. The detailed chromatographic data of the analysed samples are shown in

Table 2. LC–HRMS qualitative analysis of aqueous acetone extracts from aerial and underground parts of selected Potentilla species.

No.	Compounds	Rt (min)	UV Spectra (λ Max nm)	Observed 1	Δ (ppm)	Formula	Fragmentation		Presence in Extracts												Ref.
							Negative	Positive	PAL7r	PAU7	PER7	PER7r	PFR7	PHY7	PME7	PNE7	PPE7	PPU7	PRI7	PTH7
1	Gallic acid	5.72	270	169.01335	−3.02	C7H6O5	169, 125			+	+	+	+	+	+	+	+	+	+	+	(s)
2	2-Pyrone-4,6-dicarboxylic acid	6.82	316	182.99292	−2.56	C7H4O6	366, 183, 139	185		+	+	+	+		+	+	+	+	+	+	[24]
3	Bis-HHDP-gluconic acid	11.2	255sh	799.06359	0.27	C34H24O23	799, 497, 301								+				+
4	Unknown	13.25	310	281.02976	−1.48	C12H10O8	281, 237	283, 191, 163			+				+	+	+
5	O-Caffeoylglucaric acid isomer	15.26	298, 326	371.06060	−1.98	C15H16O11	371, 209, 191							+	+			+		+	[25]
6	Pedunculagin α or β	15.74	260sh	783.06839	−0.48	C34H24O22	783, 481, 301			+	+	+	+	+	+	+	+	+	+	+	(s)
7	Procyanidin B-type trimer	15.9	278	865.19739	−1.98	C45H38O18	865, 575, 289	867, 579, 291	+
8	Bis-HHDP-glucose	16.58	260sh	783.06730	−1.29	C34H24O22	783, 481, 301									+	+
9	O-Caffeoylglucaric acid isomer	17.75	310sh, 326	371.06162	−1.12	C15H16O11	371, 209, 191				+			+	+		+	+		+	[25]
10	5-O-caffeoylquinic acid	20.09	295sh, 325	353.08747	−0.96	C16H18O9	353, 191, 179	355, 163										+	+		(s)
11	Catechin or epicatechin O-hexoside isomer	20.35	278	451.12458	−1.55	C21H24O11	451, 289, 245	291	+				+
12	O-p-Coumaroylglucaric acid isomer	20.42	312	355.06630	−1.84	C15H16O10	355, 209, 191, 147										+	+		+	[25]
13	Dihydroxybenzoic acid O-pentoside	21.9	280	285.06146	−0.55	C12H14O8	285, 152											+		+
14	Methylgallate O-glucoside	22.01	268	345.08239	−1.59	C14H18O10	345, 183, 168	185											+	+
15	O-Caffeoylglucaric acid isomer	22.05	300, 326	371.06085	−3.3	C15H16O11	371, 209, 191							+	+		+	+			[25]
16	Galloyl-HHDP-glucose	22.26	250sh	633.07245	−0.24	C27H22O18	633, 301							+		+
17	O-p-Coumaroylglucaric acid isomer	23.12	312	355.06611	−2.36	C15H16O10	355, 209, 191, 147										+	+		+	[25]
18	Pedunculagin α or β	23.3	260sh	783.06805	−0.82	C34H24O22	783, 481, 301	303		+	+	+	+	+	+	+	+	+	+	+	(s)
19	Procyanidin B-type dimer O-hexoside	23.36	280	739.18619	−2.06	C36H36O17	739, 451, 289	741, 579, 291	+
20	Digalloyl-HHDP-gluconic acid	23.89	274	801.07970	1.57	C34H26O23	801, 633, 301, 169			+									+
21	Galloyl-HHDP-glucose	24.13	280sh	633.07357	0.4	C27H22O18	633, 481, 301			+		+	+	+	+	+	+	+	+	+
22	Catechin or epicatechin O-hexoside isomer	24.45	280	451.12352	−2.38	C21H24O11	451, 289	453, 291	+					+
23	Catechin or epicatechin C-hexoside isomer	25.22	280	451.12257	−4.09	C21H24O11	451, 289, 271	453, 291	+
24	Procyanidin A-type tetramer	25.6	280	1151.24372	−2.22	C60H48O24	1151, 863, 575, 289	1153, 865, 577, 291	+
25	Procyanidin B1	25.9	280	577.13480	−0.55	C30H26O12	577, 289	579, 289, 257	+	+		+		+			+				(s)
26	O-Feruloylglucaric acid isomer	25.95	282, 326	385.07702	−2.26	C16H18O11	385, 209, 191, 147							+			+	+			[25]
27	Procyanidin B3	26.25	280	577.13426	−1.28	C30H26O12	577, 289	579, 289, 257				+	+	+				+	+	+	(s)
28	Catechin	27.05	280	289.07096	−2.33	C15H14O6	289, 245	291, 139	+	+	+	+	+	+			+	+	+	+	(s)
29	Caffeic acid	27.55	292, 320sh	179.03455	−2.54	C9H8O4	179, 135	181								+					(s)
30	O-Feruloylglucaric acid isomer	27.98	300sh, 318	385.07654	−2.63	C16H18O11	385, 209, 191, 147							+			+	+		+	[25]
31	Digalloyl-pentose	28.02	278	453.06751	0.08	C19H18O13	453, 301				+					+
32	3-O-caffeoylquinic acid	28.35	295sh, 326	353.08691	−2.82	C16H18O9	353, 191	355, 163					+					+		+	(s)
33	Digalloyl-HHDP-glucose	28.56	275	785.08401	−0.87	C34H26O22	785, 301, 275						+						+
34	Procyanidin C2	28.87	280	865.19788	−0.51	C45H38O18	865, 575, 289	867, 579, 289	+			+	+	+						+	[23]
35	Digalloylglucose isomer	29.32	276	483.07714	−2.2	C20H20O14	483, 169, 125						+
36	Digalloylglucose isomer	29.98	278	483.07745	−1.11	C20H20O14	483, 169, 125			+			+	+		+	+
37	Laevigatin E isomer	30.2	274	1265.13990	1.26	C54H42O36	1265, 632, 301					+								+
38	Methylgalloylmalic acid	30.77	278	299.04042	−1.91	C12H12O9	299, 183, 168, 133												+
39	Quercetin O-hexoso-O-deoxyhexoso-hexoside	30.9	254, 354	771.19840	−0.79	C33H40O21	771, 609, 462, 299	773, 611, 465, 303		+
40	Laevigatin E isomer	31.07	275sh	1265.13669	−1.27	C54H42O36	1265, 632, 301							+
41	Catechin or epicatechin O-hexoside isomer	31.43	280	451.12343	−2.47	C21H24O11	451, 289	453, 291	+
42	Procyanidin B-type trimer	31.54	280	865.19810	0.01	C45H38O18	865, 575, 289	867, 579, 291	+				+	+
43	Galloyl-HHDP-glucose	31.73	272	633.07313	−0.04	C27H22O18	633, 481, 301			+	+		+	+	+	+	+	+	+	+
44	Quercetin O-hexoso- O-hexoso-pentoside	31.82	256, 354	757.18267	−0.82	C32H38O21	757, 462, 299	759, 627, 465, 303		+
45	Procyanidin B-type trimer	32.01	280	865.19703	−1.1	C45H38O18	865, 575, 289	867, 579, 291	+			+
46	Brevifolincarboxylic acid	32.32	278, 360	291.01408	−1.94	C13H8O8	291, 247	293		+	+		+	+	+	+	+	+	+	+
47	Procyanidin B2	33.71	278	577.13502	−0.04	C30H26O12	577, 289	579, 291, 139	+												(s)
48	Galloyl-HHDP-glucose	33.8	256, 342	633.07336	−0.48	C27H22O18	633, 481, 301										+			+
49	Ellagic acid derivative	33.84	280sh	898.13120	2.12	C36H35O27	898, 783, 633, 301			+				+
50	Brevifolincarboxylic acid isomer	34.01	284sh, 342	291.01448	−0.94	C13H8O8	291, 247								+					+
51	Quercetin O-hexoso- O-hexoso-pentoside	34.03	254, 342	757.18241	−0.85	C32H38O21	757, 595, 462, 299	759, 597, 465, 303											+
52	Laevigatin E isomer	34.19	275sh	1265.13618	−1.68	C54H42O36	1265, 632, 463, 301							+
53	Quercetin O-hexoso- O-uronic acid derivative	34.2	254, 346	639.11995	−0.44	C27H28O18	639, 463, 300	641, 479, 303		+								+		+
54	Procyanidin A-type tetramer	34.46	280	1151.24437	−1.65	C60H48O24	1151, 863, 575, 289	1153, 865, 577, 287	+
55	Quercetin O-diuronic acid derivative	34.5	256, 352	653.09909	−0.32	C27H26O19	653, 447, 301	655, 479, 303			+		+	+				+
56	Procyanidin B-type tetramer	34.54	280	1153.26132	−0.53	C60H50O24	1153, 576, 289	1155, 867, 577, 289				+
57	p-Coumaroylquinic acid isomer	35.14	312	337.09286	0.02	C16H18O8	337, 191	339, 147										+
58	Quercetin O-hexoso-O-hexoside	35.15	256, 352	625.14017	−1.24	C27H30O17	625, 462, 299	627, 465, 303		+						+	+		+
59	Quercetin O-hexoso-O-uronic acid derivative	35.4	254, 346	639.12105	−2.16	C27H28O18	639, 463, 301	641, 465, 303		+				+		+	+			+
60	Digalloyl-HHDP-glucose	35.47	276	785.08456	−0.06	C34H26O22	785, 615, 301, 169						+						+
61	Epicatechin	35.74	280	289.07181	0.6	C15H14O6	289, 245	291, 139	+			+									(s)
62	Kaempferol O-hexoso-O-uronic acid derivative	36	264, 338	623.12581	0.57	C27H28O17	623, 284	625, 463, 287			+
63	Quercetin O-hexoso- O-hexoso-deoxyhexoside	36.03	256, 348	771.19893	−0.45	C33H40O21	771, 462, 299	773, 627, 465, 303								+			+
64	Procyanidin B-type trimer	36.07	280	865.19912	0.04	C45H38O18	865, 575, 289	867, 579, 291				+	+
65	Caffeoylisocitric acid	36.32	300sh, 328	353.05058	−2.24	C15H14O10	353, 191, 179, 173, 155									+	+		+	+	[25]
66	Procyanidin B-type dimer	36.35	280	577.13479	−0.45	C30H26O12	577, 289	579, 291				+	+
67	Kaempferol O-hexoso-deoxyhexoso-O-uronic acid derivative	37.05	266, 346	769.18282	−0.4	C33H38O21	769, 284	771, 625, 463, 287			+
68	Dimeric hydrolysable tannin	37.55	270	1569.15737	−1.82	C68H50O44	1569, 784, 469, 301													+
69	Valoneic acid dilactone	37.6	255sh, 362	469.00441	−0.32	C21H10O13	469, 425, 301								+		+				[26]
70	Ellagic acid derivative	37.76	268, 342	741.18713	−0.79	C32H38O20	741, 579, 446, 301												+
71	Procyanidin A-type trimer	37.81	280	863.18110	−2.08	C45H36O18	863, 711, 573, 411, 289	865, 575, 287	+
72	Kaempferol O-hexoso-deoxyhexoso-deoxyhexoso- O-uronic acid derivative	37.86	266, 346	915.24077	−0.13	C39H48O25	915, 285	917, 771, 625, 463, 287			+
73	Galloyl-ellagic acid-O-hexoside	38.3	250, 374	615.06204	−1.43	C27H20O17	615, 463, 301					+		+			+	+		+
74	Quercetin O-uronic acid derivative	38.4	254, 350	725.11985	−0.75	C30H30O21	725, 505, 300	727, 479, 303		+								+		+
75	O-Caffeoylmalic acid	38.54	298, 326	295.04504	−2.45	C13H12O8	591, 295, 179, 133	295, 135									+	+		+	[27]
76	Methylgalloyl-galloyl-glucose	38.85	270	497.09317	−1.17	C21H22O14	497, 345, 183, 169												+	+
77	Sanguisorbic acid dilactone	38.88	255sh, 362	469.00439	0.38	C21H10O13	469, 425, 301								+						[26]
78	Kaempferol O-diuronic acid derivative	38.92	265, 350	637.10483	0.37	C27H26O18	637, 461, 285	639, 463, 287			+			+				+
79	Quercetin O-(malonyl- hexoso)-O-hexoside	39.08	256, 354	711.14146	−0.09	C30H32O20	711, 667, 462, 299	713, 551, 465, 303		+				+					+
80	Trigalloylglucose isomer	39.1	276	635.08854	−0.04	C27H24O18	635, 465, 313, 169						+				+		+		[28]
81	Kaempferol O-hexoso- O-hexoside	39.31	262, 348	609.14565	−1.26	C27H30O16	609, 446, 283	611, 449, 287									+			+
82	Ellagic acid O-uronic acid derivative	39.4	252, 362	477.03029	−1.24	C20H14O14	477, 301					+									[23]
83	Brevifolin	39.6	275, 350	247.02448	−1.92	C12H8O6	247, 191	249			+		+		+	+	+				[28]
84	Laevigatin isomer	39.77	255	1567.14302	−0.99	C68H48O44	1567, 783, 633, 301												+	+	[23]
85	Quercetin O-deoxyhexoso- O-deoxyhexoso-hexoside	39.8	256, 354	755.20392	−0.22	C33H40O20	755, 609, 446, 299	757, 611, 449, 303		+
86	Trigalloylglucose isomer	40.05	276	635.08863	−0.5	C27H24O18	635, 465, 313, 169						+								[28]
87	Isorhamnetin O-hexoso- O-hexoso-pentoside	40.1	254, 352	771.19887	−0.64	C33H40O21	771, 476, 315, 300	773, 641, 479, 317											+
88	Procyanidin B-type dimer O-gallate	40.28	278	729.14551	−0.59	C37H30O16	729, 577, 559, 289, 169	731, 289					+
89	Syringic acid derivative	40.44	280	313.05569	−2.4	C13H14O9	313, 197, 182												+
90	Procyanidin A-type trimer	40.52	280	863.18180	−0.75	C45H36O18	863, 573, 411, 289	865, 287	+
91	Isorhamnetin O-hexoso- O-uronic acid derivative	40.67	266, 348	653.13595	0.25	C28H30O18	653, 477, 314	655, 479, 317												+
92	Apigenin C-dihexoside	40.75	270, 332	593.15106	−0.09	C27H30O15	593, 473, 383, 353	595, 439, 355, 325		+								+		+
93	Procyanidin B-type tetramer	40.8	280	1153.25158	−0.3	C60H50O24	1153, 863, 576, 289	1155, 865, 577, 289				+
94	Procyanidin C1	41.2	280	865.19784	−0.81	C45H38O18	865, 577, 289	867, 579, 291	+												(s)
95	Ellagic acid O-uronic acid derivative	41.47	252, 360	477.03021	−0.63	C20H14O14	477, 301						+
96	Procyanidin A-type tetramer	41.51	280	1151.24448	−1.56	C60H48O24	1151, 863, 575, 289	1153, 865, 577, 287	+
97	Ellagic acid O-hexoside	41.64	252, 362	463.05127	−0.58	C20H16O13	463, 301					+	+		+	+	+	+	+	+	[23]
98	Isorhamnetin O-diuronic acid derivative	41.8	254, 352	667.11526	0.2	C28H28O19	1335, 667, 491, 315	669, 493, 317			+			+				+		+
99	Quercetin O-deoxyhexoso- O-hexoso-pentoside	42.35	254, 352	741.18832	−0.66	C32H38O20	741, 446, 299	743, 611, 449, 303		+
100	Isorhamnetin O-hexoso- O-uronic acid derivative	42.43	254, 352	653.13560	−0.3	C28H30O18	653, 477, 315	655, 479, 317								+
101	Ellagic acid O-hexoside	42.7	250, 370	463.05107	−1.61	C20H16O13	463, 301					+		+			+		+		[23]
102	Agrimonic acid A or B	43.11	270sh	1103.08618	0.64	C43H32O31	1103, 935, 783, 301, 169					+						+		+	[23]
103	Tetragalloylglucose isomer	43.13	278	787.10004	−0.98	C34H28O22	787, 465, 169						+								[28]
104	Kaempferol O-deoxyhexoso-hexoso-O-deoxyhexoside	43.16	266, 346	739.20805	−0.74	C33H40O19	739, 593, 430, 283	741, 595, 433, 287		+										+
105	Quercetin O-hexoso-O-hexoside	43.17	264, 344	625.14038	−1.14	C27H30O17	625, 463, 300	627, 465, 303									+
106	Catechin or epicatechin O-hexoside isomer	43.3	278	451.12511	0.25	C21H24O11	451, 289	289					+
107	Procyanidin B-type dimer	43.6	280	577.13537	−0.24	C30H26O12	577, 289	579, 287	+				+
108	Galloyl-bis-HHDP-glucose	44.2	255	935.08057	−0.13	C41H28O26	935, 633, 467, 301			+	+		+		+	+	+		+	+	[20]
109	Laevigatin isomer	44.6	255	1567.14331	−0.8	C68H48O44	1567, 783, 633, 301			+	+	+	+	+				+	+	+	[23]
110	Procyanidin A-type tetramer	45.03	280	1151.24657	0.26	C60H48O24	1151, 863, 575, 289	1153, 865, 577, 289	+
111	Quercetin O-hexoso-O-hexoside	45.39	254, 346	625.14019	−1.3	C27H30O17	625, 463, 300	627, 465, 303									+
112	HHDP-NHTP-glucose	45.56	254	933.06390	−0.43	C41H26O26	933, 631, 466, 301				+				+	+
113	Kaempferol O-diuronic acid derivative	45.86	266, 336	637.10484	−0.55	C27H26O18	637, 461, 285	639, 463, 287		+
114	Laevigatin isomer	45.99	255	1567.14487	0.19	C68H48O44	1567, 783, 301							+				+			[23]
115	Procyanidin B-type pentamer	46.13	278	1441.32708	1.23	C75H62O30	1441, 1153, 863, 575, 289	1443, 1155, 865, 577, 289				+
116	Quercetin O-hexoso-O-uronic acid derivative	46.5	264, 340sh	639.12041	0.17	C27H28O18	639, 463, 301	641, 465, 303									+
117	Kaempferol O-hexoso- O-hexoso-deoxyhexoside	46.63	264, 350sh	755.20300	−0.67	C33H40O20	755, 593, 447, 285	757, 595, 449, 287								+		+		+
118	Galloyl-bis-HHDP-glucose	47.7	275sh	935.07950	−0.36	C41H28O26	935, 633, 467, 301								+	+	+			+
119	Apigenin C-hexoso-C-pentoside	49.2	268, 340	563.13970	−1.45	C26H28O14	563, 519, 473, 443, 383, 353	565, 379, 355, 325												+
120	Quercetin O-hexoso-pentoside	50.08	255, 352	595.13009	0.61	C26H28O16	595, 300	597, 465, 303					+		+		+
121	Quercetin O-hexoso-pentoside	50.95	256, 354	595.12982	−0.59	C26H28O16	595, 300	597, 465, 303		+			+				+
122	Quercetin O-deoxyhexoso- O-uronic acid derivative	51.17	254, 352	623.12438	−1.13	C27H28O17	623, 301	625, 479, 303										+
123	Feruloylisocitric acid	51.46	284, 326	367.06617	−2.08	C16H16O10	367, 173									+	+			+	[25]
124	Laevigatin isomer	51.56	255	1567.14348	−0.69	C68H48O44	1567, 783, 301			+		+	+	+				+		+	[23]
125	Isorhamnetin O-hexoso-hexoside	51.65	270, 350	639.15591	−1.2	C28H32O17	639, 314, 300	641, 479, 317						+	+	+	+	+	+	+
126	Quercetin O-deoxyhexoso-hexoside	52.15	256, 354	609.11075	−0.28	C27H30O16	609, 300	611, 449, 303		+
127	Quercetin O-galloyl-hexose	52.7	264, 352	615.09845	−1.15	C28H24O16	615, 463, 300, 169	617, 303					+
128	Laevigatin isomer	52.76	255	1567.14432	−0.15	C68H48O44	1567, 783, 301					+		+			+	+		+	[23]
129	Quercetin O-pentoso-hexoside	53.41	256, 354	595.12983	−1.16	C26H28O16	595, 300	597, 435, 303											+
130	Ellagic acid O-methyl ether O-uronic acid derivative	54.1	254, 360	491.04680	0.22	C21H16O14	491, 315, 299.9					+			+		+	+		+	[23]
131	Trigalloyl-HHDP-glucose	54.3	270	937.09372	−1.14	C41H30O26	937, 783, 468, 301, 169			+				+					+
132	Ellagic acid O-pentoside	56.35	252, 360	433.04120	−0.54	C19H14O12	463, 301					+			+	+					[23]
133	Trigalloyl-HHDP-glucose	56.57	278	937.09504	−0.77	C41H30O26	937, 468, 301												+
134	Quercetin O-galloyl-hexose	57.03	264, 354	615.09882	−1.5	C28H24O16	615, 463, 300, 169	617, 303					+
135	Ellagic acid	57.7	254, 370	300.99864	−0.98	C14H6O8	301, 275	303		+	+	+	+	+	+	+	+	+	+	+	(s)
136	Isoquercitrin (Quercetin 3-O-glucoside)	59.8	254, 354	463.08790	−0.94	C21H20O12	463, 300, 271	465, 303					+		+		+	+		+	(s)
137	Tetragalloylglucose isomer	62	278	787.09906	−1.22	C34H28O22	787, 465, 169						+							+	[28]
138	Rutin (Quercetin 3-O-rutinoside)	63.7	256, 354	609.14556	−0.6	C27H30O16	609, 300, 271	611, 465, 303					+			+		+	+		(s)
139	Hyperoside (Quercetin 3-O-galactoside)	64.13	255, 355	463.08829	−0.76	C21H20O12	463, 300	465, 303		+			+	+		+	+	+	+	+	(s)
140	Kaempferol O-hexoso-pentoside	65.75	266, 348	579.13529	−0.02	C26H28O15	579, 284	581, 449, 287							+
141	Quercetin O-uronic acid derivative	66.03	256, 354	477.06730	−0.23	C21H18O13	477, 301	479, 303			+		+	+		+	+	+		+
142	Kaempferol O-hexoside	67.4	252, 350	447.09290	−1.32	C21H20O11	447, 284	449, 287		+								+
143	Kaempferol O-uronic acid derivative	68.88	254, 348	461.07221	−1.04	C21H18O12	461, 285	463, 287		+
144	Galloyl-bis-HHDP-glucose	69.66	260sh	935.07940	−0.31	C41H28O26	935, 467, 301			+		+	+	+	+	+	+	+	+	+
145	Quercetin O-pentoso-O- pentoso-uronic acid derivative	71.48	254, 352	739.17255	−0.32	C32H36O20	739, 300	741, 609, 433, 303							+
146	Dimeric ellagitannin	72.47	270	1871.16610	−0.21	C82H56O52	1871, 1265, 935, 783, 301												+
147	Kaempferol O-deoxyhexoso-hexoside	72.86	266, 345sh	579.13479	−1.17	C26H28O15	579, 284	581, 449, 287											+
148	Kaempferol O-deoxyhexoso-deoxyhexoso-O-hexoside	74.17	264, 346	739.20843	−0.33	C33H40O19	739, 593, 284	741, 595, 449, 287			+
149	Quercetin 3-O-arabinoside	85.55	254sh, 350	433.07623	−3.28	C20H18O11	433, 300	435, 303					+								(s)
150	Isorhamnetin O-pentoso-hexoside	86.15	254, 354	609.14566	−1.28	C27H30O16	609, 314, 300	611, 479, 317											+
151	Dimeric ellagitannin	87.1	250sh	1869.14746	−1.81	C82H54O52	1869, 934, 783, 301												+	+
152	Quercetin O-pentoso-deoxyhexoside	87.15	256, 352	579.13374	−3.01	C26H28O15	579, 300	581, 435, 303					+
153	Kaempferol O-deoxyhexoso-O-hexoside	88.6	264, 346	593.15030	−1.35	C27H30O15	593, 447, 284	595, 449, 287								+		+	+
154	Quercetin derivative	88.75	256, 350	607.12966	−1.56	C27H28O16	607, 300	609, 303												+
155	Astragalin (Kaempferol 3-O-glucoside)	88.8	264, 350	447.09299	−0.34	C21H20O11	447, 284	449, 287		+	+		+	+	+	+	+	+	+	+	(s)
156	Quercetin O-deoxyhexoso-hexoside	89.1	256, 348	609.14510	−1.89	C27H30O16	609, 300	611, 448, 303					+
157	Kaempferol 3-O-glucuronide	89.25	265, 350	461.07183	−1.38	C21H18O12	461, 285	463, 287						+		+	+				(s)
158	Isorhamnetin O-deoxyhexoso-deoxyhexoso-O-hexoside	89.33	254, 352	769.21962	−0.11	C34H42O20	769, 315	771, 625, 479, 317			+
159	Kaempferol O-uronic acid derivative	89.6	268, 342sh	461.07133	−2.38	C21H18O12	461, 285	463, 287					+				+
160	Kaempferol O-deoxyhexoso- O-hexoso-deoxyhexoside	89.7	266, 348	737.19349	0.28	C33H38O19	737, 593, 284	739, 593, 433, 287												+
161	Apigenin O-hexoside	90.1	266, 336	431.09795	−1.23	C21H20O10	431, 268	433, 271										+
162	Agrimoniin	90.3	250sh	1869.14917	−0.89	C82H54O52	1870, 1085, 934, 783, 301			+	+	+	+	+	+	+	+	+	+	+	(s)
163	Ellagic acid O-methyl ether O-pentoside	90.45	280sh, 365	447.05603	−1.61	C20H16O12	447, 315, 301								+						[28]
164	Kaempferol derivative	90.61	264, 348	723.17755	0.1	C32H36O19	723, 621, 579, 284	725, 593, 287							+
165	Kaempferol derivative	91.08	266, 348	635.16048	−2.02	C29H32O16	635, 284	637, 287												+
166	Apigenin O-uronic acid derivative	91.35	266, 336	445.07769	0.26	C21H18O11	891, 445, 269	447, 271		+
167	Isorhamnetin O-deoxyhexoso-hexoside	91.43	254, 352	623.16185	0.37	C28H32O16	623, 314	625, 479, 317								+			+
168	Pentagalloylglucose isomer	91.7	280	939.11106	−0.58	C41H32O26	939, 769, 469, 169						+						+
169	Isorhamnetin 3-O-glucoside	91.72	254, 350	477.10350	−0.23	C22H22O12	477, 314, 300	479, 317								+					(s)
170	Kaempferol derivative	91.94	266, 350	737.19229	−1.6	C33H38O19	737, 284	739, 593, 287												+
171	Isorhamnetin O-deoxyhexoso-hexoso O-pentoside	92.02	254sh, 355	753.18765	−0.65	C33H38O20	753, 314	755, 623, 317							+
172	Isorhamnetin O-uronic acid derivative	92.79	254, 354	491.08194	−1.69	C22H20O13	491, 315, 300	493, 317						+		+		+
173	Isorhamnetin O-pentoso- O-deoxyhexoso-O-uronic acid derivative	92.93	254, 354	767.20286	−1.49	C34H40O20	767, 621, 314	769, 623, 493, 317												+
174	Chrysoeriol O-uronic acid derivative	93.7	266sh, 346	475.08764	−0.91	C22H20O12	951, 475, 299	477, 301		+
175	Kaempferol O-acetylhexoside	94.62	264, 346	489.10342	−1.01	C23H22O12	489, 284	491, 287									+	+	+	+
176	Kaempferol derivative	94.8	266, 348	591.13497	−0.5	C27H28O15	591, 284	593, 287												+
177	Quercetin O-uronic acid derivative	94.87	266sh, 360	477.06702	−0.36	C21H18O13	477, 301	479, 303		+	+			+				+
178	Kaempferol O-malonylhexoside	94.97	266sh, 348	533.09266	−1.62	C24H22O14	533, 284	535, 287										+
179	Isorhamnetin O-galloyldeoxyhexoside	95.2	270, 348	629.11322	−2.41	C29H26O16	629, 314, 299, 169	631, 317					+
180	Naringenin O-hexoside	95.52	276sh, 362	433.11345	−1.15	C21H22O10	433, 271	435, 273									+
181	Isorhamentin derivative	95.83	254, 352	621.14502	−1.78	C28H30O16	621, 314, 300	623, 317												+
182	Isorhamnetin O-acetylhexoside	95.96	254, 352	519.11432	−0.23	C24H22O13	519, 314, 299	521, 317								+			+	+
183	Acacetin	96.26	254	283.06188	0.6	C16H12O5	283, 268	285, 242							+						[29]
184	Apigenin O-acetylhexoside	97.3	266, 326	473.10900	−0.77	C23H22O11	473, 269											+
185	Apigenin	98.1	268, 338	269.04538	−1.93	C15H10O5	269	271										+			(s)
186	Quercetin O-deoxyhexoso- deoxyhexoso-O-hexoside	98.43	266, 346	753.22397	−0.86	C34H42O19	753, 299	755, 609, 463, 301			+
187	Isorhamnetin O-hexoside	98.9	256, 356	477.10244	−3.05	C22H22O12	477, 314, 299	479, 317					+
188	Isorhamnetin O-hexoside	99.21	256, 354	477.10227	−2.92	C22H22O12	477, 314, 271	479, 317					+
189	Tricin O-deoxyhexoso- deoxyhexoso-O-hexoside	99.5	254, 354	783.23498	−0.36	C35H44O20	783, 329	785, 639, 493, 331			+
190	trans-Tiliroside	101.41	268, 315	593.13011	0.27	C30H26O13	593, 284	595, 287		+	+		+	+	+	+	+	+	+	+	(s)
191	Isorhamnetin O-pentoside	101.5	258, 354	447.09386	−1.61	C21H20O11	447, 315, 271	449, 317					+
192	Kaempferol derivative	101.87	268, 330	623.13981	−0.98	C31H28O14	623, 284	625, 287		+	+			+	+		+	+	+	+
193	cis-Tiliroside	102.37	268, 315	593.12995	−0.3	C30H26O13	593, 284	595, 287		+	+		+	+	+	+	+	+	+	+
194	Isorhamnetin O-deoxyhexoside	102.65	256, 350	461.10762	−2.95	C22H22O11	461, 314, 271	463, 317, 274					+
195	Tricin O-uronic acid derivative	103.04	254sh, 352	505.09843	−0.58	C23H22O13	505, 329	507, 331, 316			+
196	Isorhamnetin derivative	103.4	256, 350	593.14977	−2.29	C27H30O15	593, 314, 299	595, 317					+
197	N1, N5, N10-Tricoumaroyl spermidine	104.47	295, 310sh	582.26028	−0.48	C34H37N3O6	582, 462, 342, 285	584, 438, 292, 147		+	+			+	+	+	+	+	+	+	[30]
198	Ellagic acid derivative	111.73	350sh, 362	422.99970	0.41	C20H8O11	423, 343, 269							+

¹ Exact mass of [M-H]⁻ ion; sh—peak shoulder; bold—most aboundantion; (s)—reference substance; HHDP—hexahydroxydiphenoyl group; NHTP—nonahydroxytriphenoyl group.

and in Supplementary Figures S1–S12. To summarize, the number of compounds shared by all the analysed Potentilla species may typify their chemical profile as homogeneous.

2.3. 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. IC₅₀ values (concentration causing viability/proliferation inhibition by 50% compared to control) of aqueous acetone extracts isolated from selected Potentilla L. species. IC₅₀ values were calculated for human colon epithelial cell line CCD841 CoN and human colon adenocarcinoma cell line LS180 based on results of MTT as well as BrdU assays performed after 48 h of cells’ treatment with investigated compounds.

Samples	MTT Assay						BrdU Assay
	LS180			CCD841 CoN			LS180			CCD841 CoN
	IC50 (µg/mL)	Trust Range (µg/mL)	R2	IC50 (µg/mL)	Trust Range (µg/mL)	R2	IC50 (µg/mL)	Trust Range (µg/mL)	R2	IC50 (µg/mL)	Trust Range (µg/mL)	R2
PAL7r	82	77–87	0.980	496	396–623	0.908	52	41–64	0.917	412	351–483	0.841
PAU7	192	180–206	0.920	1575	536–4632	0.672	1495	1311–1704	0.871	2058	1626–2604	0.542
PER7	176	166–186	0.957	672	474–952	0.891	1001	847–1183	0.845	3705	2368–5796	0.336
PER7r	110	101–120	0.943	523	326–839	0.595	54	44–66	0.925	337	281–405	0.856
PFR7	89	85–92	0.989	707	450–1113	0.737	50	40–62	0.916	282	244–327	0.809
PHY7	156	146–167	0.952	489	334–717	0.838	425	350–516	0.843	631	495–804	0.765
PME7	128	122–133	0.983	380	291–495	0.870	417	325–536	0.774	837	661–1061	0.740
PNE7	112	106–118	0.977	1795	329–9800	0.365	343	298–395	0.915	586	451–763	0.748
PPE7	158	150–167	0.966	620	367–1047	0.663	343	283–414	0.850	911	728–1140	0.768
PPU7	197	185–210	0.967	865	359–2081	0.531	881	761–1019	0.836	937	803–1093	0.846
PRI7	213	200–228	0.968	2402	788–7326	0.717	542	452–649	0.848	1230	837–1806	0.553
PTH7	225	215–236	0.956	969	443–2119	0.643	606	521–704	0.876	1039	791–1364	0.693
5-FU	31	28–33	0.977	113	81–157	0.884	15	13–16	0.956	94	80–111	0.933

, 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 IC₅₀ 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 inflammation-induced 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 PGE₂ 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].

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 dose-dependent 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 IC₅₀ 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 IC₅₀ 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].

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].

3. Materials and Methods

3.1. Chemicals

The reference phytochemicals, including isorhamnetin 3-O-glucoside, kaempferol 3-O-glucuronide, and quercetin 3-O-glucuronide were obtained from Extrasynthese (Genay, France). Gallic acid, catechin, and epicatechin were obtained from Carl Roth (Karlsruhe, Germany). Procyanidin B1, procyanidin B2, procyanidin B3, and procyanidin C1 were purchased from Cayman Chemical (Ann Arbor, MI, USA), while agrimoniin, apigenin, 3-O-caffeoylquinic acid, ellagic acid, astragalin (kaempferol 3-O-glucoside), pedunculagin, avicularin (quercetin 3-O-arabinoside), hyperoside (quercetin 3-O-galactoside), isoquercitrin (quercetin 3-O-glucoside), and tiliroside (purity > 96%) were previously isolated in the Department of Pharmacognosy of Medical University of Białystok (Białystok, Poland) [22,48,49,50,51]. All other analytical grade chemicals used in the study were obtained from Sigma-Aldrich (St. Louis, MO, USA). To obtain ultra-pure water, a POLWATER DL3-100 Labopol (Kraków, Poland) system was used. Investigated extracts (100 mg/mL) and 5-fluorouracil (50 mM) were dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions. Working solutions were prepared by dissolving stock solutions in a culture medium. The final concentration of DMSO in all working solutions used in the studies was 0.25%.

3.2. 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. List of plants from the Potentilla genus that were screened in the study and extraction yields.

Sample Name	Lant Species	Voucher Specimen No.	Parts Used 1	Extraction Yield (%) 2
PAL7r	Potentilla alba L.	PAL-17039	R	11.2%
PAU7	Potentilla aurea L.	PAU-20045	A	32.8%
PER7	Potentilla erecta (L.) Raeusch	PER-06016	A	17.8%
PER7r			R	15.7%
PFR7	Potentilla fruticosa L. (syn. Dasiphora fruticosa (L.) Rydb.)	PFR-06018	L	36.6%
PHY7	Potentilla hyparctica Malte	PHY-20046	A	26.5%
PME7	Potentilla megalantha Takeda	PME-18043	A	34.1%
PNE7	Potentilla nepalensis Hook.	PNE-06023	A	33.4%
PPE7	Potentilla pensylvanica L.	PPS-08025	A	22.4%
PPU7	Potentilla pulcherrima Lehm.	PPU-18044	A	28%
PRI7	Potentilla rigoi Th. Wolf	PRI-20047	A	30.6%
PTH7	Potentilla thuringiaca Bernh.	PTH-06022	A	22.8%

¹ A, aerial parts; L, leaves; R, rhizomes; ² Extraction yield of purified fraction.

.

3.3. Phytochemical Profile

3.3.1. 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.

3.3.2. 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.

3.3.3. 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.

3.3.4. 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 (AlCl₃) 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.

3.3.5. 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].

3.4. 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% CO₂ at 37 °C.

3.5. 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 5-fluorouracil (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].

3.6. 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 IC₅₀ 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.

4. 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 B-type 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.