Flowers and Leaves Extracts of Stachys palustris L. Exhibit Stronger Anti-Proliferative, Antioxidant, Anti-Diabetic, and Anti-Obesity Potencies than Stems and Roots Due to More Phenolic Compounds as Revealed by UPLC-PDA-ESI-TQD-MS/MS

The present work aims to assess the biological potential of polyphenolic compounds in different parts (flowers, leaves, stems, and roots) of Stachys palustris L. Towards secondary metabolites profile, 89 polyphenolic compounds (PCs) were identified by UPLC-PDA-ESI-TQD-MS/MS, with a total average content of 6089 mg/100 g of dry matter (d.m.). In terms of biological activity, antioxidant activity (radical activity, reducing power), digestive enzyme inhibitory (α-glucosidase, α-amylase, pancreatic lipase) effect, and antiproliferative activity (inhibition of cell viability and induction of apoptosis in different human cancer cell lines) were explored. Leaves, flowers, stems, and roots of S. palustris L. have not been studied in this regard until now. Vescalagin and cocciferin d2, isoverbascoside (verbascoside), luteolin 6-C-glucoside, luteolin 6-C-galactoside, apigenin 6-C-glucoside, (−)-epicatechin, ellagic acid, and malvidin 3-O-diglucoside were detected as main ingredients in the studied parts. Methanolic extract of S. palustris L. leaves and flowers revealed the highest amount of PCs with the strongest antiradical (18.5 and 15.6 mmol Trolox equivalent (TE)/g d.m., respectively) and reducing power effects (7.3 and 5.6 mmol TE/g d.m.). Leaf extracts exhibited better α-amylase and pancreatic lipase inhibition effects, while flower extracts exhibited better α-glucosidase inhibition effect. Regarding antiproliferative activity, extracts of the leaves and flowers significantly reduced cell viability and induced a high level of apoptosis in human lung, pancreatic, bladder, and colon cancer cell lines, as well as in human acute myeloid leukemia; whereas the extracts from stems and roots revealed the weaker effects. The results of this work showed anti-proliferative and potentially anti-diabetic, anti-obesity properties of S. palustris L., especially for flowers and leaves, which may have wide potential applications in the functional food, special food, pharmaceutical, cosmetics industries, and/or in medicine.


Introduction
In recent years, edible plants, forgotten plants, wild plants, as well as medical plants have emerged as the potential sources of secondary metabolites for therapeutic interventions [1], which has opened doors for their use as active ingredients in food, pharmaceutical, cosmetics, and/or medical products.
Stachys palustris L. (sect. Stachys; family Lamiaceae, subfamily Lamioideae), otherwise known as marsh woundwort, is one such edible plant found in both lowlands and mountains, which has been used in traditional medicines, such as emetic, antiseptic, nervine, sedative, antispasmodic, emmenagogue, hemostatic, vulnerable, expectorant, and tonic agent [2][3][4][5]. However, it is most effective in treating internal and external hemorrhages, cramps, and joint pains, as well as the treatment of gout [2]. This plant can grow up to 1 m tall, has square stems, purplish-red flowers arranged in whorls, opposite leaves, and dry four-chambered schizocarp fruit [2].
The biosynthesis of polyphenolic compounds is an intensive process, largely conditional on abundant factors linked with the plant and their environment. The distribution and concentration of bioactive compounds in different underground and above-ground parts of the plant can be strongly varied. This is mainly tied to the role of polyphenols in the growth phase and in plants' life cycle [6]. Previous research has shown that S. palustris L. plants exhibit a high total polyphenol content of about 213 mg gallic acid equivalent (GAE)/g dry matter (d.m.), high antiradical activity against DPPH radical, and promising anti-proliferative properties against cervix adenocarcinoma cells (HeLa) [2,7]. On the other hand, the chromatographic analysis showed the presence of only eight compounds belonging to the group of phenylethanoid glycosides, isoscutellarein derivatives, phenolic acid, and iridoids [2].
Despite the above data and traditional knowledge about S. palustris L. being a high nutraceutical potential, little is known about the secondary metabolites profile and biological activity of edible leaves, flowers, stems, and roots of S. palustris L. [6,7]. Therefore, this study aimed to assess the profile and amount of polyphenolic compounds by UPLC-PDA-ESI-TQD-MS/MS and their in vitro biological activity (antioxidant and anti-proliferative activity). Results, on the polyphenol profile and anti-proliferative activity, as well as inhibitory activity against digestive enzymes, could be interesting for potential applications in functional foods, natural health products, pharmaceuticals, cosmetics industries, and/or medicine.

Profile of Polyphenolic Compounds
The UPLC-PDA-ESI-TQD-MS/MS fingerprint of Stachys palustris L. flowers, leaves, stems and roots (Table 1, and Figure S1) discovered the presence of 89 polyphenolic ingredients based on their UV maxima, m/z, [M-H] − and [M-H] + , retention times, peak areas, available data, and standards. Amongst identified compounds, 40 hydrolysable tannins, 16 phenylethanoid glycosides (PhGs), 4 anthocyanins, 1 flavan-3-ol, 6 phenolic acids, 1 flavonols, and 21 flavones were found. However, the qualitative composition of the tested bioactive ingredients was strongly dependent on the extracted part of the plant; with 56, 55, 50, and 49 compounds identified, respectively, in flowers, roots, leaves, and stems of S. palustris L (Tables 1 and 2). It is worth emphasizing that thus far only eight compounds had been identified in S. palustris L. belonging to secondary metabolites, such as verbascoside, echinacoside, two isoscutellarein derivatives, monomelittoside, chlorogenic acid, harpagide, and its derivative 8-O-acetyl-harpagide [2]. However, in our study, compounds, such as monomelittoside, chlorogenic acid, harpagide, and its derivative 8-O-acetyl-harpagide [2], were not observed. Thus, apart from the compounds confirmed by Venditti et al. [2], the remaining compounds were detected in the flowers, leaves, stems, and roots extracts of S. palustris L. for the first time.  a Values that are expressed as the mean (n = 3) ± standard deviation and different letters (between morphological parts) within the same row indicate statistically significant differences by Duncan's test (p < 0.05); b nd, not identified.

Flavanones
The compounds belonging the group of flavones usually subsist as glycosides and seldom as free aglycones [37]. In the full-scan LC-MS/MS, the deprotonated pseudomolecular ions [M-H] − of luteolin, apigenin, isoscutellarein, and chrysoeriol (m/z 285.0, m/z 269.0, m/z 285.0, and m/z 299, respectively) were observed [33]. A total of 21 compounds are reported in different parts of S. plasturis L., including 7 flavones in the flowers, 9 in the leaves, 8 in the stems where apigenins and luteolins dominated, and 15 in the roots where isoscutellarein and chrysoeriol dominated. The pathway for flavones C-glycosides showed the decomposition of glycan with a loss of neutral residues with the decomposition of a flavonoid moiety associated to the residual part of glycan [38]. These compounds for the genus Stachys are considered important chemosystematics markers [25].
The first class of flavones was luteolin ([M-H] − at m/z 447) determined based on characteristic UV absorption maxima at 269 and 349 nm. According to Lin et al. [37], these compounds are characterized by two UV spectral maximums: 250-300 nm (band I), and 342-350 nm (band II). Based on the fragmentation of the peak that indicated the presence of a C-glycosylated derivative, these compounds were confirmed as luteolin 6-O-galactoside and luteolin 6-O-glucoside (previously identified in the Elaeis guineensis Jacq.) [33,34].
Apart from luteolins, four apigenin compounds (m/z 431, 577, and 635) were also identified based on the UV spectrum at 260 and 336 nm. Two compounds with molecular ion at m/z 431 and the fragmentation of MS/MS indicative of a C-glycosylated derivative and loss of glucosyl (162 m.u.) moiety were identified as apigenin 6-C-galactoside and apigenin 6-Cglucoside, previously noted in Cyclanthera pedata leaves, fruits, and dietary supplements [35]. The compound with pseudomolecular ion at m/z 635 was tentatively identified on the basis of fragmentation earlier described in Stachys parviflora L. [26] as apigenin acetylallosylglucoside. Mass spectra of apigenin with p-coumaric acid derivative showed molecular ion at m/z 577 and was confirmed based on previous results for Stachys byzantina [36] and Stachys bombycina [31] as apigenin 7-O-β-D-(6-p-coumaroyl)-glucopyranoside.
Two compounds belonging to chrysoeriol derivatives with pseudomolecular ion at m/z 667 and m/z 677 were confirmed based on the fragmentation pathway described in the Stachys subgenus [32] and Stachys bombycina [31] and were identified as chrysoeriol 7-O-acetylallosylglucoside and chrysoeriol-acetyl-allopyranosyl-glucopyranoside.
Three compounds with pseudomolecular ions at m/z 756 and 637 was tentatively detected as two stachysoside A and leucoseptoside A, with a characteristic fragmentation pathway presented in Lagopsis supina [28].
The last three compounds belonging to PhGs were tentatively specified on the basis [24] noted in Phlomis species. Two compounds having pseudomolecular ions at m/z 769, and on the basis of fragmentation MS/MS the losses of p-coumaric residue, pentose, and glucose moiety, were tentatively assigned as alyssonoside and alyssonoside isomer [24]. Compounds, such as samioside (m/z 755) and martynoside (m/z 651), were assigned on the losses of glucose, pentose, rhamnose moiety, and p-coumaric and rhamnose moiety, according to the fragmentation pattern determined by Kirmizibekmes et al. [24].
2.1.4. Other Phenolic Acids, Anthocyanins, Flavonol, and Flavan-3-ol The last groups of polyphenolic compounds identified in the different parts of S. palustris L. were phenolic acids, anthocyanins, flavonol, and flavan-3-ol. Among detected phenolic acids, three compounds presented similar maximum absorbance at 325 nm, which is typical for derivatives of dicaffeoylquinic acid [25]. All of them had common pseudomolecular ions at m/z 515. The first compound was detected as 3,5-di-affeoylquinic acid compared with the standard. The next two compounds noted the characteristic ions at m/z 173, which indicate the presence of quinic acid in position four. Thus, these compounds were determined as 3,4-di-caffeoylquinic and 4,5-di-caffeoylquinic acids, respectively, on the basis of the order of elution [25]. The next three phenolic acid tentatively assigned as ellagic acid (m/z 301), ellagic acid glucoside ([M-H-463] − ), and ellagic acid pentoside ([M-H-461] − ) were previously determined in Stachys officinalis [33].

Content of Polyphenolic Compounds and Polymeric Procyanidins
Polyphenols are a very important group of secondary metabolites because they exhibit a wide range of health benefits and biological activities [41]. A statistical test on onefactor analysis indicated a significant effect (p < 0.05) of research plant parts on the total polyphenol content (TPC). The average TPC in the S. palustris L. was 6090 mg/100 g d.m. ( Table 2). The highest amount of secondary metabolites were measured in the leaves (9252 mg/100 g d.m.), and this value was 1.9 and 5.7 times higher than in the stems and roots.
A similar trend for TPC was noted in the studies on different parts of Astragalus macrocephalus subsp. finitimus [45]. In the S. palustris L. isolated from Hungary and France, the TPC was 17,630 and 24,980 mg GAE/100 g d.m., respectively [2], and was 1.8 and 2.5 times higher than in the leaves of S. palustris L. from Poland. In turn, the TPC of our extracts were significantly higher than those reported for other Stachys species (ranging from 430 mg GAE/100 g d.m. in S. trinervis to 4450 mg GAE/100 g d.m. in S. fruticulosa) from Iran by Khanavi et al. [46]. According to Oracz et al. [47], TPC may be influenced by many factors, e.g., origin, soil, weather conditions, analytical method, and the preparation of test samples. Carev et al. [42] reported that methanol extracts of S. cretica ssp. anatolica from Turkey showed 4330 mg/100 g d.m. TPC assessed by LC-ESI-MS/MS and 1.6 times lower TPC by spectrophotometric method [42]; the results were significantly lower than those reported in the flowers and leaves noted in our work. Likewise, during the analysis of S. cretica ssp. Vacillans, the record showed 3604 mg/100 g d.m. TPC using the HPLC system, and this value was 2.3 times lower compared to the spectrophotometric method [41]. On the other hand, the total concentration of TPC isolated from stems of S. officinalis L. were 6120 mg GAE/100 g dry extract [33], 1610-3330 mg GAE/100 g [48], which were 1.2 times higher and 2 times lower compared to stems of S. palustris L. The total amount of bioactive compounds extracted from leaves and roots of Stachys officinalis L. from Czech Republic were, respectively, 7495-8050 mg GAE/100 g d.m. and 2286-3164 mg caffeic acid/100 g d.m. (depending on the cultivar tested) [49]. TPC in the S. recta was 2050 mg/100 g d.m. and was around 4.8 times lower than in the leaves of S. palustris L. [25].
The most abundant group of tested parts of S. palustris L. contained a range of 1083 to 7945 mg/100 g d.m. for roots and flowers, respectively. The total tannins content identified in the different organs of Calluna vulgaris L. Hull in flowering time were 5, 4.3, and 3 times lower than our data for flowers, leaves, and stems [43]. In addition, the predominant compounds among 40 hydrolysable tannins were vescalagin and cocciferin d2 constituting 21% and 19%, respectively, in the leaves and 37% and 21% in the roots. This was also confirmed in the study by Esposito et al. [8] and Singh et al. [9].
The next quantitatively important group was flavan-3-ols, including (−)-epicatechin as a monomer (constituting 5% of all flavan-3-ols) and polymeric procyanidins (PP; constituting 95%). The average amount of (−)-epicatechin in measured parts of the plant was 12.6 mg/100 g d.m. and PP-254.3 mg/100 g d.m. The (−)-epicatechin of measured parts may be organized in the following sequence: flowers > stems > leaves, and for PP: leaves > flowers > roots > stems. A similar trend for PP was noted in the different tested organs of Rumex crispus L. and Rumex obtusifolius L. [6]. The total of catechins as catechin equivalent in Stachys marrubiifolia viv. the leaf was 40 mg/100g extract [54]. The lowest amount of flavan-3-ols was detected in the stems and also confirmed by Feduraev et al. [6], probably by the inside metabolic action of the tissues and cells and the molecular constitution of the exudate carried by the phloem channels [6]. In addition, the alkaline solution of the central cavity of stems is exposed to oxidation PCs, including flavan-3-ols [6].
Moreover, the most opulent in PhGs were the roots, and the content was 209.5 mg/100 g d.m., and this amount was an average of 34 times higher than in the rest parts of S. palustris L. The main compound detected in the roots was isoverbascoside (verbascoside). This was also confirmed in the research on S. cretica ssp. anatolica by Carev et al. [47], on S. cretica subsp. mersinaea by Bahadori et al. [41], on S. tmolea by Elfalleh et al. [50], and on Stachys cretica subsp. kutahyensis [51]. During the measurement of S. recta, the content of PhGs was 607 mg/100 g d.m. and the concentration was around three times higher than in the stems of S. palustris L. [25]. In addition, the biological activity of verbascoside was confirmed, such as, for example, anti-tumor, anti-inflammatory, anti-radical, and anti-thrombotic effects [50].
In turn, the phenolic acids were the most abundant in the flowers compared to the rest parts of S. palustris L. tested, and their amount was 2.2, 3, and 8 times higher compared to the rest fractions. The total content of phenolic acids measured in the S. recta was 709 mg/100 g d.m. [25] and was significantly higher than our results. The anthocyanins, and flavonols were noted in the flowers. These compounds present less than 1% of all PCs in the measured extract of S. palustris L. The total of anthocyanins and flavonols as quercetin and cyanidin 3-glucoside equivalent in the Stachys marrubiifolia viv. leaves were 537 and 70 mg/100 g extract [54]. According to Bahadori et al. [41], the phenolic acid content measured in S. cretica subsp. mersinaea was 401 mg/100 g extract; in S. tmolea was 118 mg/100 g [50]. In turn, anthocyanin compounds, in addition to the health benefits, are responsible for the color of the raw material. According to the analysis, the flowers are the darkest and red with the addition of dark yellow color ( Figure S1). In addition, the NAI index indicates an intense dark red pigment located in the flowers. On the other hand, the type of anthocyanins identified indicates a reddish-purple color. Of course, depending on the pH, it can change [2,29]. This is consistent with the botanical color of the flowers of the plant under study [55]. The root also seems interesting because it showed a dark red color with the addition of dark yellow [55].

Antiradical and Reducing Potential
The antioxidant properties of S. palustris L. was assessed as reducing power (FRAP assay) and radical scavenging activity (ABTS assay). Results indicate that significant differences were noted between parts of the research plant. Table 3 suggests that the flowers and leaves had the highest radical scavenging activities (18.5 and 15.6 mmol TE/g d.m. respectively) and the highest FRAP reduction potential (5.6 and 7.3 mmol TE/g d.m., respectively). The ability to scavenge synthetic ABTS radicals determined for the roots and stems was about 2 and 3.8 times and 4.4 and 4.5 times weaker compared to the flowers and the leaves. Similar conclusions were found for the iron (III) reduction capacity, which was 5.3 and 18.5 times and 4 and 14.3 times weaker compared to the flowers and the leaves. The results indicate that the flowers and the leaves are more effective at eliminating excess reactive oxygen species (ROS) that cause oxidative stress in the body compared to the roots and the stems [41,45]. When assessing the reducing power in S. anisochila, S. beckeana, S. plumosa, and S. alpina spp. Dinarica, a value of 1.9, 1.8, 0.5, and 1.4 mmol Fe 2+ /g d.m. has been reported [56]. Likewise, significantly lower values of antioxidant activity for the FRAP test were noted for the areal part of S. trinervis, S. byzantina, S. setifera, S. subaphylla, S. turcomanica, S. inflata, S. laxa, S. persica, and S. fruticulosa [45]. In S. tmolea (the areal part), the antiradical activity and reducing power was 32.3 and 41.9 mg TE/g d.m., respectively [50]. In studies conducted by Benabderrahim et al. [51], the antioxidant activity measured by ABTS and FRAP assays in S. cretica subsp. Kutahyensis were 175.8 and 239.1 mg TE/g of extract. The results obtained by Bahadori et al. [41] in Stachys cretica subsp. Mersinaea were 292.7 and 236.4 mg TE/g of extract for ABTS and FRAP tests, respectively. Whereas, in the extracts of S. cretica ssp. Anatolica for antiradical activity and reducing power were 112.2 and 127.2 mg TE/g of extract [42]. Moreover, our obtained results were not directly comparable. Besides, the research by Saravanakumar et al. [57] and Yadav et al. [58] shows that the differences in the value of antioxidant activity may significantly depend on the purity of solvents and their polarity, extraction procedure, and fractionation methods.

In Vitro Enzyme Inhibition
Digestive enzymes, such as pancreatic lipase, are involved in fat metabolism and support the digestion of dietary fats into fatty acids. A-glucosidase and α-amylase are engaged in the carbohydrate metabolism and break down complex sugars into monosaccharides and oligosaccharides [57,59]. We analyze different parts of the plant with the digestive enzyme inhibitory properties to evaluate potential anti-obesity and anti-diabetic properties ( Table 3).
The higher pancreatic lipase enzyme inhibitory activity was reported for leaves of S. palustris L. In addition, the flowers and leaves noted the higher α-amylase and αglucosidase enzyme inhibitory properties. The least ability to inhibit the activity of digestive enzymes was noted for the roots; the ability to inhibit the activity of α-amylase was on average 3.5 times weaker compared to the leaves and the flowers, while the α-glucosidase activity was 3 times weaker. The ability to inhibit pancreatic lipase activity was noted for both the roots and the flowers.
We conclude that the leaves and flowers of S. palustris L. have PP and PA compounds that show potentially high antidiabetic activities and may be used for the inhibition of these enzyme activities. To our knowledge, the inhibition of α-amylase and α-glucosidase activity had not been reported before for S. palustris L. extracts.

Anti-Proliferative Activity
Anti-proliferative activity in the leaves, flowers, stems, and roots of S. palustris L. was tested in the A549 (lung adenocarcinoma), BxPC3 (pancreatic ductal adenocarcinoma), HT-29 and CACO-2 (colorectal adenocarcinoma), HCV29T (bladder cancer), and AML-NEV007 (acute myeloid leukemia) cell lines (Figures 1 and 2). We chose tumors that are particularly resistant to chemotherapy and difficult to treat. Cells were treated with ethanol extract for 48 h, after which the MTS viability test and the analysis of the induction of apoptosis were performed.
The most significant results were obtained for leaves and flower extracts of S. palustris L. In particular, leaves extract markedly decreased the metabolic activity of all tested cell lines to 8-22%. Extract from the flower showed weaker inhibition effects (10-52% remaining activity). In addition, the roots and stems extracts were found weakest and reduced the viability to 30-90%. Diluted ethanol (1%) had no effect on cell lines.
We then tested the induction of apoptosis using Annexin V double staining and propidium iodide. As in the MTS test, the leaf and flower extracts significantly induced apoptosis in all tested cell lines in the range of 69-86%. The CACO-2 line was the most resistant, with the apoptosis level around 45%. A study by Kokhdan et al. [62] evaluated a methanol extract of S. pilifera against HT-29 cell line (colon adenocarcinoma) viability and demonstrated favorable inhibitory properties and significant anti-proliferative effects. It was also reported that S. laxa chloroform extract significantly prevented the proliferation of HT-29 and T47D (ductal carcinoma) cell lines, and the total extract of S. subaphylla also showed antiproliferative properties against T47D cell line [63]. Furthermore, Haznagy-Radnai et al. [7], noted that some Stachys species, such as S. palustris and S. recta, stems in methanolic extracts displayed significant antiproliferative activity against cervix adenocarcinoma cells (HeLa); S. germanica flowers against breast adenocarcinoma (MCF-7) cells. In addition, Lachowicz et al. [11] noted that the methanolic extract of Sanguisorba officinalis L. leaves and flowers exhibited significant antiproliferative activity versus bladder cancer (HCV29T), colorectal adenocarcinoma (DLD-1), pancreatic ductal adenocarcinoma (BxPC3), and Jurkat cell lines. The obtained extracts contain a number of different substances that may have an additive or synergistic anti-proliferative effect on cancer cells. Many of these substances affect various intracellular pathways that depend on the type of cancer cells [64]. The externalization of Annexin V indicates the activation of caspases. Most likely, the process of apoptosis follows the classical mitochondrial pathway. Polyphenolic compounds are abundant in the leaves and flowers of S. palustris L. Luteolin, ellagic acid, and apigenin derivatives are known for their activating effects on the p53 transcription factor. They also affect the cell cycle and reduce the expression of the proapoptotic proteins from the Bcl-2 family [65]. Polyphenols have anti-inflammatory properties by inhibiting the activity of COX2 and NFKB, which is important in cancer [66]. Some natural compounds have exhibited synergism with established anticancer agents, and thus may reduce the side effects of chemotherapy. Further research is needed to identify active compounds and test their anti-cancer properties in appropriate in vivo models. The above results indicate that the anti-proliferative activity is mainly influenced by the part of the plant studied and material extraction, as well as the profile of PCs.

Multivariate Analysis
The results show a significant relationship between the high value of particular groups of the PCs and enzyme inhibitory activities, antioxidant, and antiproliferative activity, which was confirmed by the Pearson correlation coefficient. It is well known that PCs indicate numerous properties, including redox properties, and they, therefore, act as singlet oxygen quenchers and hydrogen donors, as well as exhibiting antioxidant activity [50]. Moreover, it is the synergistic effect between bioactive compounds in the tested material that provides antioxidant activity depending on, among others, their concentration [50]. Thus, a very strong correlation was noted between the antioxidant potential (for ABTS and FRAP assay, respectively) and the overall value of PCs (R 2 0.953 and 0.956), as well as individual groups of substances, such as hydrolysable tannins (R 2 0.967 and 0.901), polymeric procyanidins (R 2 0.853 and 0.978), and phenolic acids (R 2 0.792 and 0.685).
In addition, anthocyanins, flavan-3-ols, and flavonols had a stronger correlation with antiradical activity than FRAP assay (R 2 0.699, 0.684, 0.699, respectively), but flavones had a stronger correlation with reducing power than ABTS assay (R 2 0.700). A strong correlation between PCs and antioxidant assays was also reported by Bahadori et al. [41,60] and Khanaki et al. [45] in research concerning the analysis of Stachys cretica subsp. Mersinaea. On the other hand, the anthocyanins, flavan-3-ols, and flavonols showed negative correlation with BxPC3 cell line, and flavones noted negative correlation with α-glucosidase inhibitory activities.
Interestingly, PhGs, especially compounds identified in the roots, were the only group of PCs to show a negative correlation with enzyme inhibitory activities, a negative strong correlation with antioxidant activity, regardless of the method used, and antiproliferative activity in the all cells line.
The strongest relationships to inhibition of α-amylase activity were noted for hydrolysable tannins, flavones, and PP against the inhibition of α-glucosidase activity-hydrolysable tannins, anthocyanins, flavan-3-ols, phenolic acid, and flavonols; whereas the strongest correlations in inhibition of pancreatic lipase activity were noted for phenolic acid and PP. In turn, α-amylase inhibitory activities indicated a positive correlation with individual PCs, apart from p-coumaric acid and TPC (R 2 −0.999) [41]. Furthermore, a strongly positive correlation was noted between the anti-proliferative potential (of all analyzed cancer cells line) and hydrolysable tannins, flavones, PP, and TPC; while the remaining groups of the polyphenolic compounds noted a positive correlation with cancer cells inhibitory activities, apart from PhGs.
Overall, the PCA results concerning different parts of plant extract data indicated a clear correlation with all PCs and anti-proliferative, anti-diabetic, anti-obesity, and antioxidant tests (Figure 3). The PCA detected two essential components that were responsible for 92.20% of data variance, including PC1 for 70.25%; while the second PC2 was only responsible for 21.94%. Figure 3 clearly shows the tested parts of S. palustris L. based on phytochemicals and antioxidant, anti-proliferative, anti-obesity, and anti-diabetic activities: stems and roots are found on the left of the plot, flowers and leaves are distributed on the right part. The obtained data indicates that the metabolites that distinguish the studied parts of the plant are the PCs and mainly hydrolyzed tannins and PP, which are found in high amounts in the leaves and flowers (PC1), and PhGs, which are especially present in the roots (PC2). Consequently, their type and concentration affect health-promoting properties. Therefore, the biological activities demonstrated a difference between parts of the plant. Besides, the difference in the profile and concentration of PCs in plant fractions is attributed to the difference in the morphological and anatomical structures and ongoing physiological processes [44,67]. The results of the PCA method showed similar results as the Pearson correlation.

Color Parameter
Color and shine of material were measured in a transmitted mode through Konica Minolta CM-700d spectrophotometer in 1 cm-thick glass trays. Measurements were conducted in CIE L*a*b* system, through a 10 • observer type and D65 illuminant [68].

Polyphenolic Compounds (PCs) by UPLC-ESI-TQD-MS/MS and Procyanidin Polymers (PP) by the Phloroglucinolysis Method
The method procedure was applied according to the protocol shared by Kapusta et al. [69]. Profiles of polyphenolic compounds were analyzed using UPLC-PDA-ESI-TQD-MS/MS. Briefly, the separation was carried out using a BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm, Waters, Warsaw, Poland) that was kept at 50 • C. For the anthocyanins investigation, the following solvent system was applied: mobile phase A (2% formic acid in water, v/v) and mobile phase B (2% formic acid in 40% ACN in water, v/v). For other polyphenolic compounds, a lower concentration of formic acid was used (0.1%, v/v). The gradient program was set, as follows: 0 min 5% B, from 0 to 8 min linear to 100% B, and from 8 to 9.5 min for washing and back to initial conditions. The injection volume of samples was 5 µL (partial loop with needle overfill), and the flow rate was 0.35 mL/min. The following parameters were used for TQD: capillary voltage 3.5 kV, cone voltage, 30 V in positive and negative mode; the source was kept at 120 • C and the desolation temperature was 350 • C, con gas flow 100 L/h, and desolation gas flow 800 L/h. Argon was used as the collision gas at a flow rate of 0.3 mL/min. The profile compounds identification was based on specific PDA spectra, mass-to-charge ratio, and fragment ions obtained after collision-induced dissociation (CID). Quantification of compounds was achieved by the injection of solutions of known concentrations that ranged from 0.05 to 5 mg/mL (R 2 ≤ 0.9998) as standards ((+)-catechin, 3,4-dicaffeoylquinic acid, luteolin 7-O-glucoside, apigenin 7-O-glucoside, kampferol-3-O-galactoside, ellagic acid, delphinidin 3-O-glucoside, cyanidin 3-O-glucoside, and malvidin 3-O-glucoside). All of the determinations were performed in duplicate and expressed as mg/L. Waters MassLynx software v.4.1 was used for data acquisition and processing. The analysis of polyphenolic compounds were assayed in triplicate, and described as mg per 100 g d.m.
The procyanidin polymers by the phloroglucinolysis method were applied according to the protocol shared by Lachowicz et al. [11]. The fractions of polymeric procyanidin were analyzed using HPLC-FL. Briefly, the separation was carried out using a BEH C18 RP column (2.1 × 5 mm, 1.7 µm; Waters Corporation, Milford, MA, USA) that was kept at 15 • C with gradient elution of solvent A as 2.5% acetic acid and solvent B as acetonitrile at a flow rate of 0.42 mL/min for a duration of 10 min (100 mm × 2.1 mm i.d., 1.7 µm, Waters) that was kept at 30 • C, and the fluorescence was recorded at the excitation and emission wavelengths at 278 and 360 nm. Quantification of compounds was made from procyanidin B2, (+)-catechin and (−)-epicatechin (R 2 ≤ 0.9995) as standards. The degree of polymerization was calculated as the molar ratio of all flavan-3-ol units (phloroglucinol adducts + terminal units) to (−)-epicatechin and (+)-catechin, which correspond to terminal units. The analysis of polymeric procyanidins were assayed in triplicate, and described as mg per 100 g d.m.

Extraction Procedure
The extraction was applied according to the protocol shared by Lachowicz et al. [11]. Briefly, 0.2 g of dry material was mixed with 7 mL of 80% of methanol in water and sonicated at 20 • C for 20 min, and then incubated at 4 • C for 24 h, and the next step was centrifuged. The supernatant was analyzed. The extractions method and all biological activities were assayed in triplicate.

Antioxidant Activity Antiradical Activity
The antiradical activity (ABTS) method was applied according to the protocol shared by Re et al. [70]. Briefly, 0.03 mL of extracted material mixed with 2.97 mL of ABTS solution and measured after 6 min at 734 nm using spectrophotometer. The result is described as mmol of Trolox equivalents (TE) per g d.m. (y = 33.64x + 2.68, R 2 = 0.998).

Reducing Potency
The reducing potency (FRAP) method was applied according to the protocol shared by Benzie and Strain [71]. Briefly, 0.1 mL of extracted material with 0.9 mL of distilled water and 3 mL of ferric complex measured after 10 min of incubation at 593 nm using spectrophotometer. The result is described as mmol of Trolox equivalents (TE) per g d.m (y = 19.82x − 1.85, R 2 = 0.999).

Ability of α-Amylase, α-Glucosidase, Pancreatic Lipase Inhibitors
Ability of α-amylase, α-glucosidase inhibitors (anti-diabetic activity), and ability of lipase activity inhibitors (anti-obesity activity) effect of the extracted material were applied according to the protocol shared by Nakai et al. [72], Podsędek et al. [73], and Nickavar et al. [74]. Briefly, potato starch solution (0.2% (v/v)), the material extract, or phosphate buffer (0.1 M; pH 6.9; control) was mixed with α-amylase, and after incubation (37 • C, 10 min) the enzymatic reaction was stopped by the addition of HCl (0.4 M), the absorbance was read at 600 nm. For the α-glucosidase assay, the material extract was mixed with enzyme solution and incubated for 10 min. After that the reaction was initiated by p-nitrophenyl-α-D-glucopyranoside solution (5 mM), and incubated (37 • C, 20 min), and read the absorbance at 405 nm. For the pancreatic lipase assay, the material extracts were mixed with enzyme solution, and incubated (37 • C, 5 min). Then, methylumbelliferone solution (0.1 mM) was added, and incubated (37 • C, 20 min) and read at an excitation wavelength of 360 nm and at an emission wavelength of 460 nm. The value of the inhibitor, required to inhibit 50% of the enzyme activity, was defined as the IC 50 value. The IC 50 of the fruits tested was obtained from the line of the plot of the fruit concentration in 1 mL of reaction mixture versus the % inhibition.

Antiproliferative Potency Cell Lines and Cell Culture
The human cancer cell lines A549 (lung adenocarcinoma), BxPC3 (pancreatic ductal adenocarcinoma), HT-29, and CACO-2 (colorectal adenocarcinoma) and HCV29T (bladder cancer) were cultured in DMEM culture medium with 10% FBS (Gibco, Thermo Fisher Scientific, Walham, MA, USA) and Antibiotic Antimycotic Solution (Sigma-Aldrich, St. Louis, MO, USA). AML-NEV007 cell line (acute myeloid leukemia) was maintained in RPMI 1640 culture medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and 10% fetal bovine serum (FBS). All cell lines were cultured at 37 • C in a humidified atmosphere of 5% CO 2 . The cells were seeded at densities of 5 × 10 3 cells/0.1 mL (0.32 cm 2 ) for cell viability assay. All cell lines were obtained from the collection of the Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.

Determination of Cell Viability
The plant extract was applied according to the protocol shared by Lachowicz et al. [11]. Cell viability was assessed by the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega), according to the manufacturer's protocol. Each treatment within a single experiment was performed in triplicate. Absorbance at 490 nm was recorded using a Wallac 1420 VICTOR2 plate reader (PerkinElmer, Waltham, MA, USA). Data were normalized to the untreated control.

Apoptosis Assay
Apoptosis was assessed by the Annexin V Apoptosis Detection Kit (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer's protocol. Briefly, the cells were incubated with all compounds for 48 h, next were stained with Annexin V-FITC (8 µg/mL) and PI (5 µg/mL) for 15 min at RT in the dark. The cells were washed with cold PBS (with Ca 2+ and Mg 2+ ) containing 2.5% FBS between the steps. Data were acquired using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed using Flowing Software 2.5.1 (Perttu Terho, Turku, Finland). Apoptosis was quantified as a percentage of both Annexin V-positive and Annexin V/PI-double-positive cells.

Statistics
Statistical analysis included post-hoc Duncan's multiple range test (p < 0.05) as oneway analysis. Principal component analysis (PCA) as the multivariate analysis were performed with Statistica 13.3 (StatSoft, Kraków, Poland).
The highest amount of PCs was detected in the leaves, followed by the flowers, stems, and roots. The strongest antioxidant activity of the ABTS and FRAP assays for the leaves and flowers extracts were exhibited. In addition, the best digestive enzyme inhibition effect as a potential antidiabetic and anti-obesity activity. One of the most important features of leaf and flower extracts is their ability to induce cell death in various tumor cell lines. In turn, in this study, the roots and stems were statistically the weakest in terms of medicinal potential.
For these reasons, S. palustris L. leaves and flowers rich in natural antioxidants with high biological activity should be further examined as health-beneficial ingredients for functional food, special food, cosmetics, and/or medical and pharmaceutical industries. Further investigations are required to isolate and identify active compounds from leaves and flowers with anti-microbiological and antiproliferative effects and a wider range of antiproliferative effects, as well as the analysis of the bioavailability of compounds.