Metabolite profiling and bioactivity of Cicerbita alpina (L.) Wallr. (Asteraceae, Cichorieae)

Cicerbita alpina (L.) Wallr. is a perennial herbaceous plant in the tribe Cichorieae (Lactuceae), Asteraceae family, distributed in the mountainous regions in Europe. In this study, we focused on the metabolite profiling and the bioactivity of C. alpina leaves and flowering heads methanol-aqueous extracts. The antioxidant activity of extracts, as well as inhibitory potential towards selected enzymes, involving in several human diseases, including metabolic syndrome (α-glucosidase, α-amylase, and lipase), Alzheimer’s disease, (cholinesterases: AChE, BchE), hyperpigmentation (tyrosinase), and cytotoxicity were assessed. The workflow comprised ultra-high-performance liquid chromatography—high-resolution mass spectrometry (UHPLC-HRMS). UHPLC-HRMS analysis revealed more than 100 secondary metabolites, including acylquinic, acyltartaric acids, flavonoids, bitter sesquiterpene lactones (STLs), such as lactucin, dihydrolactucin, their derivatives, and coumarins. Leaves showed a stronger antioxidant activity compared to flowering heads, as well as lipase (4.75 ± 0.21 mg OE/g), AchE (1.98 ± 0.02 mg GALAE/g), BchE (0.74 ± 0.06 mg GALAE/g), and tyrosinase (49.87 ± 3.19 mg KAE/g) inhibitory potential. Flowering heads showed the highest activity against α-glucosidase (1.05 ± 0.17 mmol ACAE/g) and α-amylase (0.47 ± 0.03). The obtained results highlighted C. alpina as a rich source of acylquinic, acyltartaric acids, flavonoids, and STLs with significant bioactivity, and therefore the taxon could be considered as a potential candidate for the development of health-promoting applications.


Introduction
Cicerbita alpina (L.) Wallr. (Lactuca alpina (L.) A.Gray) (alpine chicory, blue sow thistle) is a perennial herbaceous plant in the tribe Cichorieae (Lactuceae), Asteraceae family, and is distributed in the mountainous regions in Europe [1]. Commonly, its edible shoots are used as a vegetable or for salads [2]. As a vegetable, the species has a commercial value, and recently, some trials for cultivation have been performed [3,4].

Acyltartaric Acids
Similarly to acylquinic acids, a variety of acyltartaric acids (ATA) was annotated, including two mono-ATA, seven di-ATA, and three triacyltartaric acids (Table 1)  .031 → 149.009 resulting from the losses of three caffeoyl residues. This class of secondary metabolites shows a significant degree of stereoisomerism [25].
Compounds 58, 60, and 76 were identified as glycosides of isoetin (85). Isoetin is a flavone, an isobaric compound of quercetin with [M−H] − at m/z 301.035, with an additional hydroxyl group in the B ring. The structure could be evidenced by the presence of more intense RDA fragment ion 1,3 B − at m/z 149.023 (21.32%) compared to 1,3 A − at m/z 151.003 (4.27%) ( Figure 2). The 1,3 B − ion is typical for flavones, while 1,2 A − could be found in the MS/MS spectrum of the flavanols. The flavone isoetin could be distinguished from the flavonol quercetin by the presence of fragment 1,3 B − at m/z 149.023, while 1,2 A − at m/z 178.997 is characteristic of quercetin and its glycosides [27].
Compounds 74 and 86 were identified as glycosides of chrysoeriol (95) (m/z at 299.055) based on the MS/MS spectra with diagnostic fragment ions resulting from the successive loss of methyl radical •CH 3 (at m/z 284.032), CO (at m/z 256.037) and CHO (at m/z 227.034), as well as RDA ions 1,3 A − at m/z 151.003, 0,4 A − at m/z 107.012, and 1,3 B − at m/z 133.028. (Table 1) Figure S4). Compound 94 was identified as cirsiliol [19].   hydroxyl group in the B ring. The structure could be evidenced by the presence of more intense RDA fragment ion 1,3 B − at m/z 149.023 (21.32 %) compared to 1,3 A − at m/z 151.003 (4.27 %) (Figure 2). The 1,3 B − ion is typical for flavones, while 1,2 A − could be found in the MS/MS spectrum of the flavanols. The flavone isoetin could be distinguished from the flavonol quercetin by the presence of fragment 1,3 B − at m/z 149.023, while 1,2 A − at m/z 178.997 is characteristic of quercetin and its glycosides [27].

Flavanones
In the MS/MS spectra of 62, 69, and 70, loss of dihexose, hexose, and hexuronic acid, respectively, was observed; the aglycone was recorded at m/z 287.056 corresponding to eriodictyol [27]. Key fragments in the identification of these compounds were the RDA ions 1,3 B − at m/z 135.043 and 1,4 A − at m/z 125.022 (Table 1, Figure S5).
Depending on the intensity and the ratio of the fragment ions [Y0] − and [Y0-H] − , the sites for binding the sugar parts to the aglycones were also determined [29]. The identification of compounds 66-68, 72, 78-80, and 90-93 was confirmed by comparison with reference standards.

Sesquiterpene Lactones (STLs)
STL dereplication is based on fragmentation patterns and diagnostic ions in the positive ionization mode as more informative for this class of specialized metabolites [19,30]. Based on accurate mass MS spectra, MS/MS fragmentation, relative intensities of precursor and fragment ions, and elemental composition, nine guanolide STLs (96-104), derivatives of lactucin and dihydrolactucin, were tentatively identified in C. alpina extracts. Among them, three are glycosylated (96, 100, and 102) Figure S6). In addition, in negative ion a germacranolide (105), sonchuside A was identified in C. alpina leaves extract (Table 1). The identification of compounds 100 and 105 were confirmed by comparison with reference standards [8].

Coumarins
Compound 109 ([M+H] + at m/z 147.044) gave a base peak at m/z 119.049 and an intense ion at m/z 91.054 (85.17%), resulting from the sequential loss of two CO groups. Thus, the coumarin structure was proposed for 109 [31]. A similar MS/MS spectrum was obtained for 107, but here an initial loss of H 2 O was observed, resulting from the loss of the OH group, and the compound was identified as 7-hydroxycoumarin (umbelliferon) ( Table 1). By analogy, but with two hydroxyl groups, the fragmentation patterns of aesculetin (108) and aesculin (106) are explained (Table 1, Figure S7) [31].

Total Phenolic Compounds and Flavonoids Content; Antioxidant and Enzyme Inhibitory Activity
Regarding the content of total phenolic compounds, the leaves showed a higher content (75.13 ± 0.51 mg GAE/g), while a higher content of total flavonoids was observed in the flowering heads of C. alpina (Table 2). Various tests were performed to determine the antioxidant profile of the plant extracts. Tests based on different mechanisms have been used in the current work. The results are presented in Table 2. C. alpina leaves showed higher antioxidant activity in all of the used methods. The DPPH radical scavenging activity of the leaves extract was 132.80 ± 3.77 mg TE/g, and for ABTS, the value was found to be 139.54 ± 0.57 mg TE/g ( Table 2). The reducing capacity of the extracts was evaluated by CUPRAC and FRAP experiments ( Table 2). The CUPRAC method evaluated the conversion of Cu (II) to Cu (I), and FRAP indicates the reducing potential of the antioxidant, which reacts with the colorless TPTZ/Fe3 + complex to form the blue-colored TPTZ/Fe 2+ . The leaf extract has a high reducing potential (CUPRAC: 212.93 ± 11.59 mg TE/g and FRAP 141.12 ± 6.64 mg TE/g).
One of the most important mechanisms of action of antioxidants is the chelation of pro-oxidant metals. Iron is the most active metal that causes oxidative changes in cells, mainly proteins and lipids. Table 2 presented the total antioxidant activity of the extracts, assessed by the phospho-molybdenum method and the metal-chelating ability. Again, the leaves exhibited the highest total antioxidant activity (1.55 ± 0.04 mmol TE/g) and metal-chelating ability (36.97 ± 0.51 mg EDTAE/g).
The enzyme inhibitory activity of the studied extracts was determined against acetyland butyrylcholinesterase, α-amylase, α-glucosidase, and tyrosinase (Table 3). C. alpina leaves extract showed higher acetylcholinesterase (1.98 ± 0.02 mg GALAE/g) and butyrylcholinesterase inhibitory activity (0.74 ± 0.06 mg GALAE/g) ( Table 3). Flowering heads showed no butyrylcholinesterase activity. Both enzymes are considered therapeutic targets in the treatment of Alzheimer's disease. The C. alpina leaves extract also showed high activity against the enzyme tyrosinase (49.87 ± 3.19 mg KAE/g) ( Table 3). This enzyme plays a key role in the biosynthesis of melanin, being responsible for skin pigmentation. Increased melanin formation leads to skin diseases such as hyperpigmentation, skin spots, etc. Tyrosinase inhibitors are becoming increasingly important hypopigmenting agents in cosmetic and medicinal products. Regarding α-amylase and α-glucosidase inhibitory effects, the C. alpina flowering heads extract (amylase: 0.47 mmol ACAE/g and glucosidase: 1.05 mmol ACAE/g) was more active on both enzymes than leaves extract (amylase: 0.28 mmol ACAE/g and glucosidase: 0.60 mmol ACAE/g). Inhibition of these enzymes is known to be an important therapeutic strategy to control blood glucose levels in diabetic patients after a carbohydrate-rich diet. In this sense, the tested C. alpina parts could be considered as a multifunctional bioactive agent from antioxidants to enzyme inhibitors, and thus, the presented study could be valuable to provide an effective raw material in the pharmaceutical, nutraceutical, and cosmeceutical industries.

Multivariate Analysis
After the univariate analysis, eleven specialized metabolites were used to generate the PLS-DA model. PLS-DA plot demonstrated significant discrimination of both leaves and flowering heads of C. alpina ( Figure 3A). A point to note is that there is not any overlap between both extracts, and the model has performed a 100% separation ( Figure 3B). The best performance of the model was achieved for 1 component. Afterward, the importance of each bioactivity for the generating of the first component was investigated. As suggested by [32], VIPs above 1 are important and have a significant role in this model. Thus, referring to Figure 3C, all bioactivities, except acetylcholinesterase and tyrosinase, have an important role in the discrimination of the leaves and flowering heads of C. alpina. Therefore, C. alpine leaves appear to be more perspective as a result of their prominent bioactivity ( Figure 3D).
The bioactivities varied significantly within the studied C. alpina plant parts due to the presence of different metabolites in each organ responsible for the specific biological function and role in plant development, reproduction, and growth [33]. Besides, to visualize the molecules' contrast between both plant extracts, a line plot was plotted using the peak area database. Before the graphic representation, the peak area was log2 transformed. There is a great variation in the molecule levels for all the subclasses ( Figure S8). Regarding the first subclass (carboxylic, hydroxybenzoic, and hydroxycinnamic acids), salicylic acid (13) and syringic acid-O-hexoside (3) were relatively abundant in the leaves extract, while the level of quinic acid (11) was relatively higher in the flowering heads extract. Plants 2023, 12, x FOR PEER REVIEW 18 of 23 Plant polyphenols, e.g., flavonoids and phenolic acids, are multifunctional and can act as reducing agents, hydrogen-donating antioxidants, and singlet oxygen quenchers [15]. Key points in the structure of flavonoids responsible for the antioxidant activity are as follows: the o-dihydroxy structure in the B ring, the 2,3 double bond in conjugation with a 4-oxo function in the C ring, and the 3-and 5-OH groups with 4-oxo function in A and C rings, requiring for maximum radical scavenging potential. Thus, quercetin is satisfied all the above-mentioned determinants and is a more effective antioxidant than the flavanols [15] Regarding the phenolicacids, it was found that the diphenolics, chlorogenic and caffeic acids, demonstrated higher radical scavenging ability than monophenolics (p-coumaric acid), consistent with the chemical criteria applied to diphenolics. Methoxylation of the hydroxyl group in the ortho position of the diphenolic acids, as in ferulic acid, results in a decrease in the scavenging reaction, hydroxylation as in caffeic acid in place of methoxylation is substantially more effective. Ferulic acid is, indeed, expected to be more effective than p-coumaric acid due to the electron-donating methoxy group allowing increased stabilization of the resulting aryloxyl radical through electron delocalization after hydrogen donation by the hydroxyl group [15].
Previous investigation revealed that dicaffeoyl derivatives cichoric acid and 1,5dicaffeoylquinic acid demonstrated higher DPPH activity compared to monocaffeoyl derivatives, caffeoyltartaric acid (caftaric acid) and chlorogenic acid, respectively [18]. EC50 values for monocaffeoyl derivatives were found to be in the order of 20 μM, while those for dicaffeoyl derivatives had values of about 10 μM [18].
AChE activity of sesquiterpene lactones (lactucin and lactucopicrin) and different chicory extracts was previously determinated using isothermal titration calorimetry (ITC) Concerning the second subclass (hydroxycinnamic acids and derivatives), rosmarinic acid (16) was found only in the leaves. In addition, the leaves were rich in caffeoylcitramalic acid (22), while the flowering heads exhibited a relatively high concentration of caffeic acid-O-hexoside isomer (17) and caffeic acid (19). As regards the acylquinic acids subclass, four compounds, including 3-p-coumaroylquinic acid (24), 1-caffeoyl-3hydroxydihydrocaffeoylquinic acid (31), 1,3,4-tricaffeoylquinic acid (32), and 1,3-dicaffeoyl-5-hydroxydihydrocaffeoylquinic acid (40) were not presented in the flowering heads extract. However, this extract possessed a relatively high amount of several compounds i.e., neochlorogenic (3-caffeoylquinic) acid (23), 5-feruloylquinic acid (29), 3,4,5-tricaffeoylquinic acid (43), to mention only a few. In contrast, the leaf extract was rich in 1/3/5-caffeoyl-4-hydroxydihydrocaffeoylquinic acid (30). Overall, the leaf extract was relatively rich in secondary metabolites belonging to the hydroxycinnamoyltartaric acids subclass. Relating to flavones, flavonols, and flavanones, five compounds are present only in the leaves, while flowering heads have 16. However, among the metabolites presented at once in both plant organs, the flowering heads are richer in several compounds than the leaves, including Thus, the flowering heads extract is richer in flavonoids compared to the leaves but contains fewer polyphenols, e.g., acylquinic acids and displays lower bioactivity. Moreover, an antagonistic effect between some of the secondary metabolites might exist. Plant polyphenols, e.g., flavonoids and phenolic acids, are multifunctional and can act as reducing agents, hydrogen-donating antioxidants, and singlet oxygen quenchers [15]. Key points in the structure of flavonoids responsible for the antioxidant activity are as follows: the o-dihydroxy structure in the B ring, the 2,3 double bond in conjugation with a 4oxo function in the C ring, and the 3-and 5-OH groups with 4-oxo function in A and C rings, requiring for maximum radical scavenging potential. Thus, quercetin is satisfied all the above-mentioned determinants and is a more effective antioxidant than the flavanols [15] Regarding the phenolicacids, it was found that the diphenolics, chlorogenic and caffeic acids, demonstrated higher radical scavenging ability than monophenolics (p-coumaric acid), consistent with the chemical criteria applied to diphenolics. Methoxylation of the hydroxyl group in the ortho position of the diphenolic acids, as in ferulic acid, results in a decrease in the scavenging reaction, hydroxylation as in caffeic acid in place of methoxylation is substantially more effective. Ferulic acid is, indeed, expected to be more effective than p-coumaric acid due to the electron-donating methoxy group allowing increased stabilization of the resulting aryloxyl radical through electron delocalization after hydrogen donation by the hydroxyl group [15].
Previous investigation revealed that dicaffeoyl derivatives cichoric acid and 1,5dicaffeoylquinic acid demonstrated higher DPPH activity compared to monocaffeoyl derivatives, caffeoyltartaric acid (caftaric acid) and chlorogenic acid, respectively [18]. EC50 values for monocaffeoyl derivatives were found to be in the order of 20 µM, while those for dicaffeoyl derivatives had values of about 10 µM [18].
AChE activity of sesquiterpene lactones (lactucin and lactucopicrin) and different chicory extracts was previously determinated using isothermal titration calorimetry (ITC) and docking simulation. The results showed strong interactions of STLs as well as extracts from chicory with AChE. In a test of enzyme activity inhibition after introducing acetylcholine into the model system with STL, a stronger ability to inhibit the hydrolysis of the neurotransmitter was observed for lactucopicrin, which is one of the dominant STL in chicory. The inhibition of enzyme activity was more efficient in the case of extracts [34].
The presented study revealed 110 secondary metabolites, including 13 carboxylic, hydroxybenzoic acids, and their glycosides, 9 hydroxycinnamic acids, and derivatives, 21 acylquinic acids, 12 acyltartaric acids, 40 flavones, flavonols, and flavanones, 5 coumarins and 10 sesquiterpene lactones. Ninety-five of all annotated compounds are reported for the first time in C. alpina. Our results for the total flavonoid content could be compared to those obtained for the wild collection of alpine chicory by Alexandru et al. [4], while the total phenolic content is significantly higher than their result. Oppositely, the data for the edible shoots of cultivated C. alpina are prominently higher compared to our data.

Cytotoxicity Assay
To evaluate the C. alpina cytotoxicity, a human monocytic cell line (THP-1 cells) mimics the behavior of the extracts toward the immune system was used (Figure 4). After 24 h of incubation of macrophage cell line THP-1 with flowering heads and leaves extracts, a slight rise of metabolic activity is measured between 2 and 200 µg.mL −1 . We reach toxicity of 70% for 2000 and 3000 µg.mL −1 for flowerings head extracts, and of almost 90% for leaves, at the same concentration. The latter concentrations are very high and could be considered meaningless.
ics the behavior of the extracts toward the immune system was used (Figure 4). Afte of incubation of macrophage cell line THP-1 with flowering heads and leaves extra slight rise of metabolic activity is measured between 2 and 200 µ g.mL −1 . We reach to of 70% for 2000 and 3000 µ g.mL −1 for flowerings head extracts, and of almost 90% leaves, at the same concentration. The latter concentrations are very high and cou considered meaningless.   flow rate 12; spare gas flow rate 0; capillary temperature 320 • C; probe heater temperature 320 • C; S-lens RF level 50; scan mode: full MS (resolution 70,000) and MS/MS (17,500). The chromatographic separation was performed on a reversed-phase column Kromasil EternityXT C18 (1.8 µm, 2.1 × 100 mm) at 40 • C. The chromatographic analyses were run using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) as a mobile phase. The flow rate was 0.3 mL/min. The run time was 33 min. The following gradient elution program was used: 0-1 min, 0-5% B; 1-20 min, 5-30% B; 20-25 min, 30-50% B; 25-30 min, 50-70% B; 30-33 min, 70-95%; 33-34 min 95-5%B. Equilibration time was 4 min [19]. Data were processed by Xcalibur 4.2 (Thermo Scientific) instrument control/data handling software. Metabolite profiling using MZmine 2 software was applied to the UHPLC-HRMS raw files of the studied C. alpina extracts.

Total Phenolic and Flavonoid Content
Total phenols and flavonoids were evaluated as gallic acid (GAE) and rutin (RE) equivalents, respectively, using spectrophotometric methods. The experiments were performed as previously reported [37,38].

Determination of Antioxidant and Enzyme Inhibitory Activities
Extracts antioxidant and enzyme inhibitory effects (0.2-1 mg/mL) were evaluated using spectrophotometric assays. Detailed protocols were reported elsewhere [19,39].

Cytotoxicity Assay
THP-1 cells (at 1.10 5 cells/mL) cells in RPMI medium (Thermo-Fisher) supplemented with 10% FBS were seeded in each well of a 48-well plate (n = 4). Cells were permitted to adhere for 24 h, and then treated with leaves and flowering heads C. alpina extracts in the medium for 24 h. Then 40 µL of WST-1 testing solutions (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and the plate incubated at 37 • C for 2 h. The contents of each well were laid down in 3 wells of a 96-well plate. The absorbances were measured at 350 and 630 nm with an Omega StarLab spectrophotometer (Omega, Ortenberg, Germany).

Statistical Analysis
In the antioxidant and enzyme inhibitory assays, the values are expressed as mean ± SD of three parallel experiments. In terms of antioxidant and enzyme inhibitory abilities, the student t-test (α = 0.05) was performed to determine differences between the tested extracts. The statistical analysis was performed using XlStat 16.0 software. Clustered image maps (CIM) were used to visualize metabolite variation among the extracts. Prior to CIM analyses, data were normalized and centered. Afterward, supervised partial least-square discriminant analysis (PLS-DA) was performed to discriminate the different parts regarding their biological activities. Then CIM was applied to PLS-DA outcomes to characterize each extract. Lastly, Pearson's correlation coefficients were calculated to evaluate the relationship between secondary metabolites and biological activities, respectively.

Conclusions
More than 100 secondary metabolites, including carboxylic, hydroxybenzoic, hydroxycinnamic, acylquinic, and acyltartaric acids, flavones, flavonols, flavanones, sesquiterpene lactones, coumarins, and their derivatives were annotated/dereplicated in the C. alpina leaves and flowering heads. Ninety-five, including acylquinic acids, acyltartaric acids, and flavonoids, were reported for the first time in C. alpina. Cichoric, caftaric, and chlorogenic acids dominated in the leaves, while apigenin, 3,5-di, and 3,4-dicaffeoylquinic acids dominated in the flowering heads profiling. The connection between the different plant parts and biological activity was performed using multivariate statistical analyses. The pronounced antioxidant activity (DDPH, FRAP, CUPRAC, ABTS, Chelating, and Phosphomolibdenum capacity) and enzyme inhibitory potential against AChE, BChE, tyrosinase, and lipase of the leaves extract could be related to the higher content of total polyphenols and the presence of acyltartaric and monoacylquinic acids compare to flowering heads. The prominent α-glucosidase and α-amylase inhibitory activity of the flowering heads correspond to the higher level of total flavonoids, luteolin, apigenin, and their glycosides. The studied extracts expressed low cytotoxicity towards THP-1 viability. In addition to inducing an antioxidant response, C. alpina extracts displayed enzyme inhibitory effects in vitro, which generates interest in the plant as a potential candidate for attenuating metabolic-related disorders. Moreover, this study supports further investigation towards the additional in vivo studies and corroborates the application of C. alpina in the pharmaceutical and nutraceutical industries.