Orbitrap Mass Spectrometry-Based Profiling of Secondary Metabolites in Two Unexplored Eminium Species and Bioactivity Potential

The study aimed at the metabolite profiling and evaluation of antioxidant and enzyme inhibitory properties of methanol extracts from flowers, leaves, and tubers of unexplored Eminium intortum (Banks & Sol.) Kuntze and E. spiculatum (Blume) Schott (Araceae). A total of 83 metabolites, including 19 phenolic acids, 46 flavonoids, 11 amino, and 7 fatty acids were identified by UHPLC-HRMS in the studied extracts for the first time. E. intortum flower and leaf extracts had the highest total phenolic and flavonoid contents (50.82 ± 0.71 mg GAE/g and 65.08 ± 0.38 RE/g, respectively). Significant radical scavenging activity (32.20 ± 1.26 and 54.34 ± 0.53 mg TE/g for DPPH and ABTS) and reducing power (88.27 ± 1.49 and 33.13 ± 0.68 mg TE/g for CUPRAC and FRAP) were observed in leaf extracts. E. intortum flowers showed the maximum anticholinesterase activity (2.72 ± 0.03 mg GALAE/g). E. spiculatum leaves and tubers exhibited the highest inhibition towards α-glucosidase (0.99 ± 0.02 ACAE/g) and tirosinase (50.73 ± 2.29 mg KAE/g), respectively. A multivariate analysis revealed that O-hydroxycinnamoylglycosyl-C-flavonoid glycosides mostly accounted for the discrimination of both species. Thus, E. intortum and E. spiculatum can be considered as potential candidates for designing functional ingredients in the pharmaceutical and nutraceutical industries.


Introduction
The search for bioactive natural compounds derived from medicinal plants is of great importance in modern medicine [1]. Natural products have been a rich source of pharmacologically active molecules that have been used to develop numerous drugs [2]. Furthermore, plant-derived compounds have been shown to have diverse biological activities, including antimicrobial, anti-inflammatory, and anticancer effects [3,4]. These compounds can serve as lead molecules for the development of new drugs or as alternative treatments to synthetic pharmaceuticals. Additionally, the use of natural products can help to reduce the potential side effects associated with synthetic drugs. In light of the growing interest in natural products, it is crucial to continue exploring the vast potential of medicinal plants to identify new and effective bioactive compounds that can benefit human health. Values are reported as mean ± SD of three parallel measurements. GAE: Gallic acid equivalent; RE: Rutin equivalent. Different letters indicate significant differences between the tested extracts (p < 0.05).

UHPLC-HRMS (Orbitrap) of Specialized Metabolites in Eminium Extracts
To estimate the specialized metabolites, non-targeted metabolic profiling of the carboxilic, phenolic, amino, and fatty acids, their derivatives, and flavonoids of both E. intorum and E. spiculatum extracts was carried out by UHPLC-Orbitrap-HRMS. Based on the MS and MS/MS accurate masses, fragmentation patterns, retention times, and comparison with reference standards and the literature data, a total of 83 metabolites, including 19 phenolic acids and derivatives, 46 flavonoids, 11 amino acids and derivatives, and 7 fatty acids were identified or tentatively annotated in Eminium extracts (Table 2).        Carboxylic, Hydroxybenzoic, Hydroxycinnamic, Acylquinic Acids, and Saccharides  [20].

Amino Acids and Derivatives
Based on the comparison with the literature data, two amino acids (71 and 73), a dipeptide (74), and six amino acid hexosides were tentatively annotated ( Table 2) Thus, 74 was related to γ-glutamyl-leucine [19]. MS/MS spectra of compounds 66, 67, 68, 69, 70, and 72 demonstrated base peaks, resulting from the loss of a hexose moiety, with the appearance of the corresponding amino acid residue. Hence, they were annotated as hexosides of glutamic acid, valine, tyrosine, leucine, phenylalanine, and tryptophan, respectively (Table 2) [19].

Antioxidant Activity
To study the antioxidant activity of E. intortum and E. spiculatum methanolic extracts, we used six different methods including DPPH, ABTS cation, FRAP, CUPRAC, phosphomolybdenum, and metal chelate assays. As shown in Table 3, all the extracts tested showed significant radical scavenging activity. The methanolic extracts of E. intortum exhibited a much stronger antioxidant activity compared to the extracts of E. spiculatum, with values of 32.20 ± 1.26 mg TE/g for leaves, 27.87 ± 0.75 mg TE/g for flowers, and 26.90 ± 1.61 mg TE/g for tubers. The methanolic extract of the tuber of E. spiculatum also showed good antioxidant activity with a value of 33.67 ± 1.26 mg TE/g. Table 3 illustrates the ability of the extracts to scavenge the ABTS cation. The methanolic fractions of all studied parts of both plants had similar significant scavenging activity against ABTS. According to the CUPRAC method, the methanolic extract of the leaves of both plants showed significant antioxidant activity, with a value of 88.27 ± 1.49 for E. intortum and 86.27 ± 2.74 for E. spiculatum. The methanolic extracts of the tubers and flowers of both plants showed lower antioxidant activity compared to the leaves. In the FRAP assay, a high absorbance indicates a high reducing power. The methanolic extracts of the tubers of both E. spiculatum and E. intortum showed very significant reducing activity, with values of 41.33 ± 1.25 mg TE/g and 32.58 ± 4.02 mg TE/g, respectively. The other extracts of both plants also showed significant results of antioxidant activity in this assay. In summary, our findings suggest that the extracts are rich in phenolic compounds with radical-scavenging activity and proton-donating ability. Polyphenols have been acknowledged for their antioxidant activity, which may account for their potential ability to prevent various diseases associated with oxidative stress. This assertion is supported by several studies [27][28][29][30]. When using the phosphomolybdenum method, all plant extracts displayed low levels of antioxidant activity with comparable values, as this method is specific in nature. Conversely, when using the metal chelation method, significant effects were observed in the methanolic extracts of the leaves of both plants, with values of 63.43 ± 0.70 mg EDTAE/g for E. intortum and 61.55 ± 3.97 mg EDTAE/g for E. spiculatum. It should be noted that this method is also highly specific.
The antioxidant activity of a substance is influenced by how it interacts with radicals in the reaction medium. These interactions are facilitated by active molecules that trap the radicals, and previous studies have highlighted their importance [31][32][33]. The efficacy of antioxidants is not solely dependent on the concentration of the main constituents but also on the presence of other constituents in smaller amounts, or the synergy between the constituents [34][35][36][37].
Limited literature exists regarding the antioxidant properties of the Eminium genus. Alkofahi et al. [11] conducted a study demonstrating the protective effect of the E. spiculatum extract against oxidative DNA damage using the 8-hydroxydeoxyguanosine assay, while no beneficial effect on alleviating oxidative damage was observed. Al-Ismail et al. [38] reported the antioxidant properties of the E. spiculatum ethanolic extract using DPPH, FRAP, and vegetable oil emulsion systems, where the extract exhibited significant properties with lower IC 50 values. Additionally, Janat and Al-Thnaibat [39] reported that the methanolic extract of E. spiculatum exhibited higher antioxidant activity in the phosphomolybdenum assay when compared to the aqueous extract.

α-Amylase and α-Glucosidase Inhibition
In the intestinal tract, the breakdown of complex sugars into simple sugars is facilitated by two essential enzymes, namely α-amylase and α-glucosidase. The simple products, particularly glucose, are subsequently absorbed and can cause a rise in blood sugar levels. To manage diabetes, one potential therapeutic approach is to inhibit the enzymes responsible for carbohydrate hydrolysis, which can decrease postprandial blood sugar levels [40,41]. By inhibiting these enzymes in the intestinal tract, the degradation of complex sugars into simple sugars can be prevented, leading to a reduction in blood sugar levels [42][43][44]. In our study, we investigated the potential inhibition of these enzymes by extracts from the two plants. Table 4 shows the inhibitory effects of methanolic extracts from various parts of the two plants on α-amylase and α-glucosidase. Our results indicate that these extracts have the potential to inhibit the activity of both enzymes. Interestingly, the methanolic extracts of leaves and flowers from E. spiculatum also exhibited α-glucosidase inhibition, with values of 0.99 ± 0.02 ACAE/g and 0.89 ± 0.01 ACAE/g, respectively. Among E. intortum extracts, the leaf extract was only active against α-glucosidase (0.35 mmol ACAE/g). In addition, both tuber extracts showed no inhibitory effect on α-glucosidase. Our results demonstrate the potential of the studied leaf extracts to act as inhibitors of carbohydrate hydrolyzing enzymes. By delaying the absorption of dietary carbohydrates in the small intestine and reducing postprandial hyperglycemia, these inhibitors may be a valuable component in the development of antidiabetic drugs. This mechanism of action has been previously reported [45][46][47]. Our study is the first to investigate the antidiabetic activity of the two plants, and we found that all studied extracts of the plants inhibited α-amylase activity, with values lower than 0.31 ± 0.01 ACAE/g. The inhibitory effect of phenolic compounds on carbohydrate hydrolysis enzymes has been reported previously [48][49][50], and this may be due to their ability to bind to proteins.

Cholinesterase Inhibition
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are enzymes found primarily in nerve tissues and neuromuscular junctions. They are responsible for rapidly hydrolyzing acetylcholine, a neurotransmitter, into inert choline and acetate. Overexpression or excessive catalysis of AChE can cause neuronal disturbances and lead to neurological disorders. A potential strategy for neuroprotection is to inhibit AChE activity [51][52][53]. Our research aimed to investigate the potential inhibitory effect of our plant extracts on the enzymes AChE and BChE. The results of this study are presented in Table 3, and they show that the extracts were able to inhibit both AChE and BChE. Specifically, the methanolic extract of both plants exhibited significant inhibitory activity against AChE, with values ranging from 1.27 ± 0.09 mg GALAE/g to 2.72 ± 0.03 mg GALAE/g. Interestingly, most of the extracts displayed greater inhibitory power on AChE than on BChE. It is worth noting that cholinesterase inhibitors are known for their therapeutic action in inhibiting acetylcholinesterase at the central level, and our findings suggest that our plant extracts could potentially be used for this purpose. Numerous studies have reported that various plant species possess the ability to block and inhibit both types of enzymes (AChE and BChE) [54][55][56].

Tyrosinase Inhibition
In skin cells, tyrosinase plays a crucial role in the aging process, and inhibiting its activity is a key strategy for delaying skin aging. This enzyme catalyzes the initial two steps of melanogenesis, making it a rate-limiting factor in this process. Mutations in the tyrosinase gene or its absence can cause a reduction or cessation of pigmentation [57,58].
In our study, we tested all the extracts against tyrosinase and demonstrated that they all exhibit activity, with variation depending on the part studied and the solvent used. The results are presented in Table 3. The methanolic extracts of all three parts of each plant showed very strong tyrosinase inhibitory activity. However, the methanolic extract of E. spiculatum and E. intortum tubers exhibited strong inhibitory activity with values of 50.73 ± 2.29 KAE/g and 48.13 ± 0.24 KAE/g, respectively. Natural compounds capable of inhibiting tyrosinase activity are of great interest, with increasing demand in the fields of cosmetics and pharmaceuticals [59,60]. Numerous studies have revealed the tyrosinase inhibitory activity of several plant species, which have demonstrated a significant effect against this enzyme [61][62][63][64][65].

Data Analysis
To gain more insight into the tested extracts, we evaluated the results of chemical components and bioactivity assays using multivariate analysis. In recent years, multivariate analysis has become increasingly popular in phytochemical studies as it helps establish connections between different parameters. By determining a small number of principal components based on Kaiser's rule, we performed PCA analysis using three components. The first component (PC1) was mainly associated with antioxidant assays and AChE. The second component (PC2) contained amylase, BChE, and tyrosinase, while the third component (PC3) included glucosidase and DPPH ( Figure 3B). Although the distribution of the samples on the score plot generated from the three principal components exhibited variability, it was challenging to clearly distinguish between different homogenous groups ( Figure 3A). A multivariate analysis has provided more information about the connections between chemical profiles and biological properties, as documented in the literature. To classify the samples and identify the biological activities that characterize each cluster, we created a heat map. Based on the heat map, we obtained four clusters. The tuber samples were classified in the same cluster (Cluster I), while Cluster II contained the flowers and leaves of E. spiculatum. In contrast, the flower and leaves of E. intortum were grouped into Cluster III and IV, respectively ( Figure 3C).
The classifications of the tested extracts based on their chemical components is presented in Figure 4. Cluster I included the leaves and flowers of E. spiculatum, which contained high levels of chrysoeriol O-coumaroylhexoside-8-C-hexoside, luteolin-6,8-Cdiglucoside, and apigenin 7-O-synapoylhexosyl-8-C-hexoside. The leaves and flowers of E. intortum were classified in Cluster II, which were characterized by high levels of luteolin 7-O-coumaroylhexosyl-6-C-hexoside and apigenin O-feruloylhexosyl-8-C-hexoside. Cluster III contained both tuber samples. Furthermore, we conducted a correlation analysis between the biological activities and individual components, and the results are shown in Figure 5. Some compounds were found to be strongly correlated with the tested biological activities. For instance, apigenin-6-C-hexoside-8-C-pentoside was the main contributor to DPPH scavenging ability, while sinapic acid-O-dihexoside and isorhamnetin-3-O-rutinoside were positively correlated with ABTS and CUPRAC. Consistent with our findings, the compounds have been described as important antioxidants by several researchers [66][67][68]. Additionally, rutin was found to be the primary player in AChE and BChE assays, consistent with previous studies [69].
DPPH scavenging ability, while sinapic acid-O-dihexoside and isorhamnetin-3-O side were positively correlated with ABTS and CUPRAC. Consistent with our fi the compounds have been described as important antioxidants by several research 68]. Additionally, rutin was found to be the primary player in AChE and BChE consistent with previous studies [69].

Plant Materials
In  16 E). The plant specimens were identified by one of our co-authors, Dr. Ugur Cakilcioglu, and one specimen from the plants was deposited at the Harran University herbarium. Prior to extraction, the plant materials were carefully washed with tap and distilled water to eliminate any soil and contaminants. After being air-dried for 10 days (in shade at room temperature), the flowers, leaves, and roots were powdered.

Extraction of Samples
For extraction, we employed the maceration method, in which 5 g of plant material was mixed with 100 mL of methanol and left to macerate at room temperature for 24 h. After maceration, the extracts were filtered through Whatman filter paper, and the solvents were evaporated using a rotary-evaporator. To preserve the extracts, we stored them at 4 • C until analysis.

Total Quantification of Phenolics and Flavonoids
We determined the total phenolic and flavonoid content of the extracts using the Folin-Ciocalteu and AlCl3 assays, respectively, according to Zengin and Aktumsek's protocol (2014). The results of these tests were reported in terms of gallic acid equivalents (mg GAE/g dry extract) and rutin equivalents (mg RE/g dry extract) [70].

Assays for Antioxidant and Enzyme Inhibition
We analyzed the extracts for a range of antioxidant and enzyme inhibitory activities, including, cupric reducing antioxidant capacity (CUPRAC), DPPH, and ABTS radical scavenging, metal chelating activity (MCA), ferric reducing antioxidant power (FRAP), phosphomolybdenum (PBD), and inhibition of amylase, tyrosinase, glucosidase, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). We employed the previously described methods to evaluate these activities [71]. Each sample was analyzed three times.

Data Analysis
All data were given as mean ± standard deviation (SD). Statistical analysis was performed by analysis of variance (ANOVA). A post hoc test (Tukey) was performed when the differences shown by data were significant (p < 0.05). Then, Principal Component Analysis (PCA) and hierarchical clustered analysis (HCA) were performed to emphasize the distinct clusters in terms of their bioactivities. Furthermore, hierarchical clustered analysis (HCA) was performed to assess the (dis)similarity between samples in terms of their molecules. All used data were scale and molecules data and were log transformed before doing multivariate analysis. R v.4.2.3 statistical program was used for all analyses.

Conclusions
In our study, we explored the detailed chemical composition and biological effects of two Eminium species: E. intortum and E. spiculatum. We found that the chemical composition and biological properties (antioxidant and enzyme inhibitory effects) varied according to the plant parts used. Generally, the leaf extract of both species exhibited higher antioxidant effects compared to flowers and tubers. However, we obtained different results for each enzyme inhibition assay, and the leaf extracts provided good anti-amylase and anti-glucosidase actions. The extracts were rich in flavonoids. These findings provide a valuable scientific basis for evaluating the potential of the Eminium genus and suggest that the tested species could be considered as a source of natural bioactive agents for health-promoting applications. Nevertheless, additional research is necessary to elucidate the potential toxicity of both the extracts and the individual chemical constituents.