Based on retention times, MS and MS/MS accurate masses and relative ion abundance, elemental composition, fragmentation patterns in MS/MS spectra, conformity to the simulated monoisotopic profiles and comparison with reference standards and literature data, a total of 61 specialized natural products were identified or tentatively annotated in C. appendiculatum extracts (Table 1). The total ion chromatograms (TIC) of the studied extracts are depicted in Figure 2.

In the biochemometric approach, peak areas (log (peak area)) for a data quantitative analysis were used. A designed graph clearly shows the differences of the phytochemical components’ distribution in the studied herbal extracts (Figure S1). Thus, flower heads contained more flavonoids compared to aerial parts and roots. Subsequently, a qualitative analysis was carried out.

Polyphenols and their biological properties are one of the most attractive topics in the natural sciences. Nowadays, humanity needs to substitute synthetic compounds with natural ones. This means safe and alternative raw materials need to be found [27]. In this sense, total phenolic and flavonoid content is considered a first insight in evaluating plant extracts. Thus, the total amount of these biocompounds in tested extracts were determined by using spectrophotometric methods (Table 2). Root extract was found to have the highest content of phenolics (143.62 mgGAE/g), followed by flower heads and aerial parts (71.75 ± 1.47 mgGAE/g and 26.02 ± 1.49 mgGAE/g, respectively).

Regarding total flavonoids, the values are in the following order: flower heads (46.59 mg RE/g) > roots (3.99 mg RE/g) > aerial parts (2.64 mg RE/g). Hence, flavonoids represent about 50% of the total phenolic components in flower head extract. According to a literature survey, different levels of total bioactive compounds in Cirsium species were observed [28,29]. These differences could be linked to the habitat of the studied plant, climate conditions or extraction procedures/solvents. However, in past years, the utilization of spectrophotometric methods for total content of bioactive compounds has led to some concerns, and these methods are not used by most scientists anymore [30]. Thus, plant matrices are very complexed and phenolics as well as other components such as peptides could be reacting with Folin’s reagent. Finally, the exact quantity of bioactive constituents has to be confirmed by chromatographic techniques such as LC–MS/MS, NMR and Q–TOF-MS analysis.

In the present study, C. appendiculatum extracts were tested for antioxidant potential (Table 2). DPPH^• and ABTS^•+ were used to evaluate radical scavenging ability. The root (97.95 mg TE/g for DPPH^• and 224.59 mg TE/g for ABTS^•+) and flower head extracts (101.79 mg TE/g for DPPH^• and 224.57 mg TE/g for ABTS^•+) displayed the strongest abilities. The aerial parts extract had the lowest capacity in both scavenging assays. The reduction abilities of the studied herbal extracts were evaluated using the CUPRAC and FRAP methods, and they are closely linked to the electrohern-donating potential of the extracts. The most prominent reduction ability was observed in the roots, followed by the flower heads and aerial parts. In terms of reduction of Mo (VI) in the phosphomolybdenum (PHMD) assay, the extracts can be ranked as follow: roots > flower heads > aerial parts. In general, the antioxidant data showed the same trends in total phenolic levels. This fact was supported by several authors who reported a strong correlation between total phenolics and radical scavenging and reducing abilities [31]. However, the metal chelating method based on the binding of transition metals by phytochemicals did not correlate with the other antioxidant methods. Regarding the metal chelating assay, the best ability was registered in the flower head extract (32.53 mg EDTAE/g), while the root sample was not active.

Nowadays people are battling noncommunicable illnesses like diabetes mellitus, obesity and Alzheimer’s. In particular, changes in lifestyle and dietary preferences increase the risk of these diseases. In the course of scientific study, some enzymes can be valuable tools against these health problems [32]. This approach is known as the enzyme inhibitory theory, in which some enzymes play a role in the pathologies of these diseases. For example, amylase and glucosidase are the main targets for controlling blood sugar levels in diabetes patients [33]. In addition, lipase is the main target for controlling obesity. In the present study, the inhibitory effects of different enzyme classes were investigated. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) belong to the same structural class of proteins, the esterase/lipase family, amylase and glucosidase are hydrolases, while tyrosinase is an oxidoreductase enzyme. Given this information, several compounds have been chemically produced as inhibitors. Although they are accepted as effective agents in the control of global health problems, concerns have been raised regarding some disturbances to wellbeing [34]. In this regard, plants are considered to be the most important and richest natural source of enzyme inhibitors such as alkaloids, phenolic acids and terpenoids. Recent studies have shown that some plants and their constituents showed promising inhibitory effects on key enzymes that have been linked to significant health problems [35]. Hence, the enzyme inhibitory properties of C. appendiculatum extracts were examined (Table 3). In both AChE and BChE inhibition assays, the root extract exhibited the highest inhibitory values (4.93 mg GALAE/g and 3.80 mg GALAE/g, respectively). The lowest abilities were recorded for aerial parts and flower head samples. Regarding tyrosinase inhibition ability, all extracts showed inhibitory effects, and the values ranged from 97.78 to 127.99 mg KAE/g in the following order: flower heads < aerial parts < roots. All the tested herbal extracts had similar amylase inhibition capacity (p > 0.05), while the strongest glucosidase ability was observed in root extract (0.72 ± 0.07 mmolACAE/g). In addition, the aerial parts sample was not active in glucosidase.

The data could be related to the different chemical components which were identified in the tested extracts (Table 1). For example, flavonoids and acylquinic acids dominate in the chemical profiles. Several flavonoids, including quercetin, luteolin and apigenin have been described as significant enzyme inhibitors [35]. In addition, chlorogenic acid and its derivatives are known to be important neuroprotectors and antidiabetic agents. Some data on the enzyme inhibitory properties of Cirsium species have been found [29]. Thus, the obtained data in the current study can be a valuable contribution to the development of new active substances agents against Alzheimer’s disease, and diabetes and its complications. The methoxylated flavone derivatives pectolinarin and its aglycon pectolinarigenin are important for the pharmacological activity of the genus [8]. The aforementioned are responsible for the control of diabetes and other metabolic disorders, and are prominently represented in the chemical composition of C. japonicum [36]. Pectolinarin and its aglycone individually significantly reduce glucose levels, but the strongest antidiabetic effect is achieved when they are combined [36].