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Article

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

by
Ebru Yuce Babacan
1,
Dimitrina Zheleva-Dimitrova
2,*,
Reneta Gevrenova
2,
Abdelhakim Bouyahya
3,
Mehmet Maruf Balos
4,
Ugur Cakilcioglu
1,
Kouadio Ibrahime Sinan
5 and
Gokhan Zengin
5
1
Pertek Sakine Genç Vocational School, Munzur University, Pertek, Tunceli 62500, Turkey
2
Department of Pharmacognosy, Faculty of Pharmacy, Medical University-Sofia, 1000 Sofia, Bulgaria
3
Laboratory of Human Pathologies Biology, Department of Biology, Faculty of Sciences, Mohammed V University in Rabat, Rabat 10106, Morocco
4
Şanlıurfa Provincial Directorate of National Education, Karaköprü, Şanlıurfa 63320, Turkey
5
Physiology and Biochemistry Research Laboratory, Department of Biology, Science Faculty, Selcuk University, University Campus, Konya 42130, Turkey
*
Author to whom correspondence should be addressed.
Plants 2023, 12(12), 2252; https://doi.org/10.3390/plants12122252
Submission received: 24 April 2023 / Revised: 1 June 2023 / Accepted: 4 June 2023 / Published: 8 June 2023
(This article belongs to the Special Issue Phytochemical Analysis and Metabolic Profiling in Plants)
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Abstract

:
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.

1. 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.
Eminium (Blume) Schott is a genus of the Araceae family, which includes over 300 species that grow mostly in tropical areas [5]. In Turkey, the Araceae family is represented by the main genera of Arum, Biarum, and Eminium. The genus Eminium includes seven taxa worldwide, five of which are distributed in Turkey, where its members are known locally as kardi, asalan, and kurtkulağı [6]. Studies on the biological activity and phytochemical constituents of Eminium are limited. Ethnobotanical records show that Eminium species are used for various purposes, including treating gastrointestinal problems [7], internal diseases, dysentery, and abdominal pain [8], yet there is a scarcity of phytochemical studies on the genus. In an earlier study by Afifi and Abu-Dahab [9], E. spiculatum (Blume) Schott was found to possess antimicrobial, antiplatelet, and antiproliferative properties, and the isolation of luteolin, luteolin-7-O-glucoside, chrysoeriol-7-O-glucoside, vitexin flowering, and isoorientin, as well as β-sitosterol from aerial parts of E. spiculatum was reported. Another study by Obeidat [10] reported antimicrobial properties of E. spiculatum against several strains of bacteria and fungi. Alkofahi et al. [11] reported the DNA protecting ability of the ethanolic fraction of E. spiculatum. To the best of our knowledge, there is no study on the chemical composition and bioactivity of E. intortum (Banks & Sol.) Kuntze.
The potential of the phytochemical screening strategy has been significantly increased by the recent progress of hyphenated techniques, which are able to afford efficient separation of the specialized metabolites and their dereplication at the same time. By coupling HPLC with mass spectrometry (LC/MS), especially high-resolution MS, a large amount of data can be obtained on the secondary metabolites of the plants’ extract before starting any isolation work [12].
Recently, ultra-high-performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS) has been introduced and become widespread in phytochemistry. HRMS is one of the most sensitive methods for analysis. Moreover, it has the potential to obtain information on the molecular weight, as well as on the structure of the secondary metabolites in plants. Due to its high potential of mass separation, very high selectivity can be obtained [12]. Thus, UHPLC-HRMS is a very suitable technique for analysis of unexplored plant species.
In light of the results of the literature review, we discovered a gap regarding the chemical profiles and biological activities of members of the genus Eminium. Therefore, we aimed to evaluate the biological properties of the methanolic extracts from three parts (leaves, flowers, and tubers) of two Eminium species (E. intortum and E. spiculatum). With regard to chemical profiles, the extracts were characterized by the UHPLC-HRMS (Orbitrap) technique. The results obtained could shed light on the biopharmaceutical potential of the member of the genus Eminium.

2. Results and Discussion

2.1. Total Phenol and Total Flavonoid Contents

The contents of total polyphenols (TP) and total flavonoids (TF) were measured for two plants, E. intortum and E. spiculatum, and the results are presented in Table 1. The three extracts from the different parts of both plants contained significant amounts of TP and TF, but the levels varied among the different plant parts and species studied. E. intortum was found to have higher levels of polyphenols and flavonoids, with the flower extract having the highest TP content (50.82 ± 0.71 mg GAE/g), followed by the leaves (25.86 ± 0.58 mg GAE/g) and tubers (21.31 ± 0.33 mg GAE/g). For flavonoids, E. intortum still had higher levels, with the leaf, flower, and tuber extracts containing 65.08 ± 0.38 mg GAE/g, 39.38 ± 0.65 mg GAE/g, and 9.81 ± 0.05 mg GAE/g, respectively.
Despite both plants belonging to the same genus and being harvested from the same places, E. intortum showed higher levels of TP and TF compared to E. spiculatum. This suggests that the differences in the chemical composition of these two plants are likely due to genetic factors.
Numerous studies have shown that the synthesis of polyphenols and flavonoids is regulated by genetics [13,14,15]. The flowers and leaves are also known to be the primary sites of storage and photosynthesis for these compounds [16,17,18]. This suggests that the high levels of TP and TF observed in E. intortum and E. spiculatum are likely due to their resistance to secondary metabolites, which is primarily achieved through the synthesis and storage of these compounds in leaves and flowers. In contrast, other parts of the plant, such as the tubers, typically have lower levels of polyphenols and flavonoids.

2.2. 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

Compound 1 [M-H]− at m/z 341.109, corresponding to two ester bond hexoses gave fragment ions at m/z 179.055 [M-H-165.05]−, due to the loss of a hexosyl moiety. Fragment ions at m/z 119.033, 89.022, and 59.012 resulting from the sugar cross ring cleavages 0,4 Hex (−60 Da), 0,3 Hex (−90 Da), and 0,2 Hex (−120 Da) were registered, respectively. Thus, compound 1 was ascribed to sucrose [19]. Compound 2 [M-H]− at m/z 191.018 showed fragment ions at 173.008 [M-H-H2O]−, 147.028 [M-H-CO2]−, a base peak at m/z 111.07 [M-H-CO2-2H2O]−, and was related to citric acid [20].
Three hydroxycinnamic acids (12, 13, 18, and 19) together with chlorogenic acid (9) were identified in the Eminium extracts on the base of comparison with reference standards (Table 2). Six hydroxybenzoic glycosides were tentatively identified including hexosides (3, 4, 5, 16, 17) along with dihexoside of sinapic acid (15). In addition, hexosides of caffeic (6), coumaric (11), and ferulic acids (14), as well as their dihexosides (7, 8, 10) were annotated based on the diagnostic ions, corresponding to the loss of one (−162.05 Da) or two (−2 × 162.05 Da) hexoses (Table 2).

2.3. Flavonoids

Thirteen mono-, twenty-nine di-, and one triglycosides, as well as three aglycons were identified/annotated in the studied Eminium extracts (Table 2). Among them, forty were flavones, five flavonols, and one flavanone. The flavone aglycone apigenin (63), luteolin (64), and chrysoeriol (65) were deduced from the Retro-Diels-Alder (RDA) rearrangements 1,3 A−, 0,4 A−, 1,2A−, 1,3 B−, and 1,2 B− (Table 2). Additionally, a series of neutral losses of CO2 (−44), CH2O (−30), CO (−28), and H2O (−18) supported the aglycone identification [21].

2.4. Flavonoid O-Glycosides

Peak dereplication of flavonoid O-glycosides was assigned by the neutral mass losses of 162.053, 308.112, and 324.106 Da consistent with hexose, rutinose/neohesperidose, and dihexose [22]. Thus, compounds 42, 43, 52, 55, 57, and 58 were ascribed to O-hexosides of quercetin, luteolin, kaempferol, apigenin, chrysoeriol, and cirsiliol, respectively. Similarly, compounds 35, 47, 50, and 56 were related to O-rutinosides of quercetin, kaempferol, isorhamnetin, and chrysoeriol. Compound 46 [M-H]− at m/z 623.162 gave a base peak at m/z 299.056, corresponding to the concomitant loss of two hexoses, and was annotated as chrysoeriol 7-O-dihexoside. MS/MS spectrum of 54 [M-H]− at m/z 577.1568 showed fragment ions at m/z 431.098 [M-H-dHex]− and 413.088 [M-H-dHex-H2O]−, and a base peak at m/z 269.045 [M-H-dHex-Hex]−. The absence of the interglycosidic linkage breakdown is favored for 7-neohesperidoside. Thus, 54 was tentatively identified as apigenin 7-O-neohesperidoside (Table 2). 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 [23].

2.5. Flavonoid C-Glycosides

Key points in the dereplication of C-glycosides in the negative ion mode are fragment ions [(M-H)-120]−, [(M-H)-90]−, and [(M-H)-30]−. C-8 isomers were characterized by the base peak 0,2 X− [(M-H)-120]− as MS/MS of orientin (30), while C-6 isomers showed the base peaks [M-H]− as homoorientin (27), isovitexin (37), and chrysoeriol 6-C-hexoside (44), together with abundant fragment 0,2 X− [(M-H)-120]− (about 60%). In addition, the fragment ion 0,3 X− [M-H-90]− was favored by C-6 isomers (Table 2) [24]. Compounds 20 and 22 gave indicative ions at m/z 519.113 and 503.119 [(M-H)-90]−, 489.103, and 473.109 [(M-H)-120]−, 429.082 and 413.089 [(M-H)-120-60]−, 399.072 and 383.077 [(M-H)-120-90]−, 369.061 and 353.066 [(M-H)-2 × 120]−, respectively. Based on 1,3 B− fragments at m/z 133.028 (20) and 117.033 (22), 20 and 22 were tentatively annotated as 6,8-C-diglucosides of luteolin and apigenin, respectively (Table 2) [24].

2.6. Flavonoid C,O-Diglycosides

Among three isobars indicated as 20, 21, and 23 with [M-H]− at 609.147, 21 and 23 were assigned to luteolin C, O-diglucosides. Typical ions of the C, O-flavon pathway resulted from the concomitant losses of hexosyl moiety (X0) and 0,3 X1 (−90) or 0,2 X1 (−120) at m/z 357.062 and 327.051, respectively (Table 2). In the same manner, 28 was ascribed as chrysoeriol C, O-diglucoside. Chrysoeriol was deduced from the prominent fragment ions at m/z 327.051 Y0−/0,2 X1/CH2, 299.0542 [chrysoeriol-H]−, 298.048 Y0−/0,2 X1/CH2/CHO, 297.041 Y0−/0,2 X1/·CH3/CO, and 269.046 Y0/0,2 X1/·CH3/2CO. Apigenin O-pentosyl-6-C-hexoside (33) was discernable by the fragment ions at m/z 341.067 ([(M-H)-132-90]− and 311.0561 ([(M-H)-132-120]−, suggesting the presence of both O-pentosyl (X1) and C-hexosyl (X0) moieties. Additionally, the aforementioned structure was assigned on the basis of a series of fragment ions at 283.061 (Y1−/0,2 X0/CO), 282.055 (Y1−/0,2 X0/CHO), 281.045 (Y1−/0,2 X0/CH2O), and 237.055 (Y1−/0,2 X0/2CO) (Table 2).
MS/MS spectrum of 29 with ([M-H]− at m/z 771.179 was acquired. In (-) ESI mode, 29 yielded prominent fragment ions at m/z 447.093 [(M-H)-2 × 162.05]−, 357.062 [(M-H)-2 × 162.05-90]−, 327.051 ([(M-H)-2 × 162.05-120]−, suggesting the presence of both O-dihexosyl and C-hexosyl moieties. The absence of the interglycosidic linkage breakdown is favored for O-sophoroside. Thus, 29 was annotated as luteolin O-sophoroside-6-C-hexosyl-orientin. Compound 32 ([M-H]− at m/z 593.151) yielded prominent ions at m/z 413.088 ([(M-H)-(162.05 + H2O)]−, 353.067 ([(M-H)-(162.05 + H2O)-60]−, 311.056 ([(M-H)-(162.05)-120]−, 293.047 [(M-H)-(162.05 + H2O)-120]−, and 281.045 Y1−/0,2 X0/CH2O indicating an O-hexosyl unit at the 2”of the primary hexose [24]. Thus, 32 was assigned to 2″-O-hexosyl-6-C-hexosyl-apigenin (Figure 1).
Two compounds 34 ([M-H]− at m/z 815.205) and 45 ([M-H]− at m/z 799.210) shared the same fragmentation patterns yielding prominent ions at m/z 447.094 (34) and 431.098 (35), respectively, indicating a concomitant loss of hexose (162.05 Da) and sinapoyl residue (206 Da, C11H10O4) (Table 2). Moreover, sinapoyl moiety was evidenced by the fragment ions at m/z 205.049 [(sinapic acid-H)-H2O]−, 190.027 [(sinapic acid-H)-H2O-CH3]−, and 175.003 [(sinapic acid-H)-H2O-2·CH3]−. The presence of a C-hexosyl moiety on the flavonoid skeleton was deduced from the relative abundances of the ions at m/z 327.051 [M-H-120]− (79.7%) (34) and 311.056 (100%) (45) suggesting a 6-C- and an 8-C-linkage, respectively. Thus, 34 was ascribed as luteolin O-sinapoylhexosyl-6-C-hexoside, while 45 was annotated as apigenin O-sinapoylhexosyl-6-C-hexoside (Figure 2).
Two caffeoyl esters 36 and 41 were discernable by the transition [M-H]−→Y0 resulting from the indicative loss of 324.085 Da (C15H16O8). Precursor ions gave a series of fragment ions involved in the C-glycosyl flavon pattern: m/z 353.064 (Y0−/0,4 X1/H2O), 341.067 (Y0−/0,3 X1), 311.056 (Y0−/0,2 X1), and 283.061 (Y0−/0,2 X1/CO) (Table 2). Caffeoyl residue was evidenced by the diagnostic ions at m/z 161.023 [(caffeic acid-H)-H2O]− and 133.028[(caffeic acid-H)-H2O-CO]−. Accordingly, 36 and 41 were assigned to apigenin O-caffeoylhexosyl-8-C-hexoside and chrysoeriol O-caffeoylhexosyl-8-C-hexoside (Figure 2).
Feruloyl esters of luteolin-, apigenin-, and chrysoeriol O-hexosyl-8-C-hexoside (38, 48, and 49) were deduced from the loss of feruloylhexosyl residue (338 Da, C16H18O8) and the subsequent transition of m/z 447.094/431.099/461.109→327.051/311.056/341.067, respectively, arising from the hexose cross ring cleavage (0,2 X1) (Table 2). The ions at m/z 175.039 [(ferulic acid-H)-H2O]−, 160.015 [(ferulic acid-H)-CH3]−, and [(ferulic acid-H)-H2O-CH3-CO]− point out to the feruloyl moiety (Table 2) (Figure 2).
In the same way, 39, 48, and 53 were ascribed as coumaroyl esters of luteolin-, apigenin-, and chrysoeriol O-hexosyl-C-hexoside. The commonly found loss of 308 Da (C15H16O7) in the fragmentation patterns of the aforementioned compounds, accompanied with the ions at m/z 163.039 [p-coumaric acid-H]−, 145.028 [(p-coumaric acid-H)-H2O]−, and 117.033 [(p-coumaric acid-H)-H2O-CO]− confirmed the presence of a coumaroyl moiety (Figure 2). Two coumaroyl esters of luteolin- and apigenin-O-deoxyhexosyl-6-C-hexoside (60 and 62) were evidenced on the base of the loss of 292 Da (C15H16O6) (Table 2) (Figure 1).
The identification of flavonoids 25, 27, 30, 35, 37, 42, 47, 50, 52, 55, 63, and 64 was confirmed by comparison with reference standards.

2.7. 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). Compound 71 [M-H]− at m/z 164.071 gave a base peak at m/z 147.043 [M-H-NH3]−, and prominent ions at m/z 146.059 [M-H-H2O]−, 119.048 [M-H-NH3-CO]−, and 103.918 [M-H-NH3-CO-CH3]−. Thus, 71 was assigned as phenylalanine [19]. Compound 73 with the molecular formula C11H12O2N2 showed fragments at m/z 186.054 [M-H-NH3]−, 159.091 [M-H-NH3-CO2]−, 142.064 [M-H-2NH3-CO2]−, and a base peak at m/z 116.049 [M-H-2NH3-CO2-C2H2]−. Thus, 73 was annotated as tryptophan; an essential amino acid previously described in the Araceae family [19,25]. MS/MS spectrum of 74 (C11H20O5N2), a series of fragment ions resulting from neutral losses were registered at m/z 241.119 [M-H-H2O]−, 223.108 [M-H-2H2O]−, 215.140 [M-H-CO2]−, 197.1287 [M-H-H2O-CO2]−, and a base peak at m/z 128.033, corresponding to the loss of leucine [M-H-131.096]− from the glutamyl residue. 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].

2.8. Fatty Acids

Based on the comparison of accurate masses and MS/MS fragmentation patterns with the literature data, one saturated (82), three monounsaturated (76, 79, and 80), and five polyunsaturated (75, 77, 78, 81, and 83) free fatty acids were tentatively annotated in Eminium extracts (Table 2) [25]. Among them, five (78–82) were dihydroxylated and three (75–77) contained three hydroxyl groups (Table 2). Compound 83 was dereplicated as linoleic acid, previously found in E. rauwolffii [26].

3. 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 IC50 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.

4. Enzymes Inhibitory Activities

4.1. 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.

4.1.1. 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].

4.1.2. 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].

4.1.3. 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-C-diglucoside, 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].

5. Materials and Methods

5.1. Plant Materials

In the summer of 2021, we collected Eminium species in Turkey (Eminium intortum (Banks & Sol.) Kuntze: Between Kızıltepe and Mardin, 533 m, GPS: 37°14′20″ N, 40°38′10″ E; Eminium spiculatum (Blume) Schott: Derik, Mardin, 661 m, GPS: 37°18′44″ N, 40°16′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.

5.2. 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.

5.3. Chemicals

Acetonitrile (hypergrade for LC–MS), formic acid (for LC–MS) and methanol (analytical grade) were purchased from Merck (Merck, Bulgaria). The reference standards used for compound identification were obtained from Extrasynthese (Genay, France) for p-coumaric, o-coumaric, ferulic acids, saponarin, homoorientin, orientin, rutin, isovitexin, isoquercitrin, luteolin 7-O-glucoside, kaempferol 3-O-rutinoside, kaempferol 3-O-glucoside, isorhamnetin 3-O-glucoside, apigenin 7-O-glucoside, quercetin, and apigenin. Chlorogenic and caffeic acids were supplied from Phytolab (Vestenbergsgreuth, Bavaria, Germany).

5.4. 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].

5.5. UHPLC-HRMS (Orbitrap)

The UHPLC-HRMS analyses were carried out on a Q Exactive Plus mass spectrometer (ThermoFisher Scientific, Inc.) equipped with a heated electrospray ionization (HESI-II) probe (ThermoScientific). The equipment was operated in negative and positive ion modes within the m/z range from 100 to 1000. The mass spectrometer parameters were as follows: spray voltage 3.5 kV (+) and 2.5 kV (−); sheath gas flow rate 38; auxiliary gas 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 achieved on a reversed phase column Kromasil EternityXT C18 (1.8 µm, 2.1 × 100 mm) at 40 °C. The UHPLC analyses were run with a mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The run time was 33 min. The flow rate was 0.3 mL/min. The gradient elution program was used as follows: 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 [22]. Data were processed by Xcalibur 4.2 (ThermoScientific, Waltham, MA, USA) instrument control/data handling software. Metabolite profiling using MZmine 2 software was applied to the UHPLC–HRMS raw files of the studied extracts. The areas under the curve (AUC) for each identified compound were plotted and used for further statistical analysis in 3.10 [22].

5.6. 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.

5.7. 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.

6. 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.

Author Contributions

Conceptualization, E.Y.B., D.Z.-D., R.G. and G.Z.; methodology, D.Z.-D., R.G., K.I.S. and G.Z.; software, E.Y.B. and G.Z.; validation, E.Y.B., A.B., M.M.B. and U.C.; formal analysis, G.Z.; investigation, E.Y.B., A.B., U.C. and G.Z.; resources, E.Y.B., U.C. and M.M.B.; data curation, G.Z.; writing—original draft preparation, E.Y.B., D.Z.-D., R.G., A.B. and G.Z.; writing—review and editing, G.Z.; visualization, D.Z.-D. and G.Z.; supervision, G.Z.; project administration, D.Z.-D. and R.G.; funding acquisition, D.Z.-D. and R.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Not applicable.

Acknowledgments

This study is financed by the European Union-NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria, project No. BG-RRP-2.004-0004-C01.

Conflicts of Interest

The authors declare no conflict of interest.

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Plants 12 02252 g001 550
Figure 1. Possible fragmentation pathway of (A) apigenin 2″-O-hexosyl-6-C-hexoside (32) and (B) apigenin 2″-O-coumaroyl-6-C-hexoside (61).
Figure 1. Possible fragmentation pathway of (A) apigenin 2″-O-hexosyl-6-C-hexoside (32) and (B) apigenin 2″-O-coumaroyl-6-C-hexoside (61).
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Plants 12 02252 g002 550
Figure 2. (-) ESI/MS-MS spectrum of apigenin O-caffeoylhexosyl-8-C-hexoside (36) (A), luteolin 7-O-coumaroylhexosyl-6-C-hexoside (39) (B), apigenin 7-O-synapoylhexosyl-8-C-hexoside (45) (C), chrysoeriol O-feruloylhexoside-8-C-hexoside (49) (D); CA-caffeic acid; p-CoA-p-coumaric acid; SA-sinapic acid; FA-ferulic acid.
Figure 2. (-) ESI/MS-MS spectrum of apigenin O-caffeoylhexosyl-8-C-hexoside (36) (A), luteolin 7-O-coumaroylhexosyl-6-C-hexoside (39) (B), apigenin 7-O-synapoylhexosyl-8-C-hexoside (45) (C), chrysoeriol O-feruloylhexoside-8-C-hexoside (49) (D); CA-caffeic acid; p-CoA-p-coumaric acid; SA-sinapic acid; FA-ferulic acid.
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Plants 12 02252 g003 550
Figure 3. Exploratory Principal Component Analysis. (A). Scatter plot showing the distribution of the samples in the factorial plan derived from the three retained dimensions. (B). Loading plots showing the relationship of biological activities on each dimension of PCA. (C). Clustered Image Map on biological activities dataset. (Blue color: low bioactivity. Red color: High bioactivity).
Figure 3. Exploratory Principal Component Analysis. (A). Scatter plot showing the distribution of the samples in the factorial plan derived from the three retained dimensions. (B). Loading plots showing the relationship of biological activities on each dimension of PCA. (C). Clustered Image Map on biological activities dataset. (Blue color: low bioactivity. Red color: High bioactivity).
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Plants 12 02252 g004 550
Figure 4. Global overview of the secondary metabolites’ contrasts among Eminium samples (Blue color: low content. Red color: high content). For compound numbers refer to Table 1.
Figure 4. Global overview of the secondary metabolites’ contrasts among Eminium samples (Blue color: low content. Red color: high content). For compound numbers refer to Table 1.
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Plants 12 02252 g005 550
Figure 5. Relationship between polyphenol metabolites and antioxidant activities of samples. For compouns numbers refer to Table 1.
Figure 5. Relationship between polyphenol metabolites and antioxidant activities of samples. For compouns numbers refer to Table 1.
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Table
Table 1. Total phenolic (TP) and flavonoid (TF) content of the tested extracts.
Table 1. Total phenolic (TP) and flavonoid (TF) content of the tested extracts.
SpeciesPartsTP (mg GAE/g)TF (mg RE/g)
Eminium intortumLeaves25.86 ± 0.58 b65.08 ± 0.38 a
Flowers50.82 ± 0.71 a39.38 ± 0.65 b
Tubers21.31 ± 0.33 c9.81 ± 0.05 c
Eminium spiculatumLeaves23.90 ± 0.57 a46.54 ± 0.40 a
Flowers21.30 ± 1.25 b19.91 ± 0.15 b
Tubers18.68 ± 0.34 c3.93 ± 0.12 c
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).
Table
Table 2. Secondary metabolites in Eminium extracts.
Table 2. Secondary metabolites in Eminium extracts.
№Identified/Tentatively Annotated CompoundMolecular FormulaExact Mass
[M-H]−		Fragmentation Pattern in (-) ESI-MS/MStR
(min)		Δ ppmDistribution
Carboxylic, hydroxybenzoic, hydroxycinnamic, acylquinic acids, and saccharides
1.sucroseC12H22O11341.1089341.1089 (100), 179.0550 (46.7), 161.0444 (12.4), 143.0337 (10.1), 131.0336 (5.7), 119.0335 (36.7), 101.0228 (39.3), 89.0228 (91.1), 71.0122 (75.6), 59.0122 (57.5)0.68−0.0431,2,3,4,5,6
2.citric acidC6H8O7191.0189191.0189 (9.6), 173.0080 (1.5), 147.0287 (0.5), 129.0179 (5.9), 111.0072 (100), 87.0071 (47.8),0.91−4.4281,2,3,4,5,6
3.salicylic acid O-hexosideC13H16O8299.0779137.0230 (100), 93.0330 (63.1)1.282.0371,2,3,4,5,6
4.protocatechuic acid-O-hexosideC13H16O9315.0723315.0723 (100), 153.0181 (25.7), 152.0103 (56.8), 109.0289 (7.3), 108.0201 (81.9), 123.0071 (3.9) 1.640.4591,2,3,4,5,6
5.salicylic acid O-hexoside isomerC13H16O8299.0772299.0772 (100), 239.0557 (20.5), 209.0449 (3.1), 179.0339 (40.4), 137.0230 (97.6)2.04−0.1021,2,3,4,5,6
6.caffeic acid-O hexoside
O-caffeoylhexose		C15H18O9341.0877341.0877 (27.6), 281.0659 (1.8), 251.0557 (0.9), 221.0447 (1.6), 161.0231 (100), 135.0438 (6.4)2.60−0.2791,2,3,4,5,6
7.caffeic acid O-dihexosideC21H28O14503.1412503.1412 (72.8), 179.0341 (15.9), 161.0232 (100), 135.0437 (9.3), 133.0281 (31.4)2.761.7521,2,3,4,5
8.caffeic acid O-dihexoside isomerC21H28O14503.1415503.1415 (100), 341.0888 (3.5), 323.0760 (9.3), 281.0680 (16.9), 251.0563 (9.9), 221.0443 (4.1), 179.0338 (27.3), 161.0231 (80.5), 135.0437 (25.8), 133.0280 (23.3)3.141.0761,2,3,4,5,6
9.chlorogenic (5-caffeoylquinic) acid aC16H18O9353.0896353.0896 (4.9), 191.0552 (100), 179.0340 (1.0)3.194.9981,2,3,4,5,6
10.ferulic acid O-dihexosideC22H30O14517.1566517.1566 (39.5), 193.0497 (100), 175.0390 (65.8), 149.0596 (4.7), 160.0153 (52.2), 134.0359 (29.9)3.260.6601,3,6
11.coumaric acid-O-hexosideC15H18O8325.0930325.0930 (13.3), 265.0714 (2.2), 235.0605 (0.4), 205.0500 (1.7), 163.0388 (12.9), 145.0282 (100), 119.0487 (4.7), 117.0331 (18.3)3.310.4901,2,3,4,5,6
12.p-coumaric acid aC9H8O3163.0387163.0387 (80.35), 135.0437 (100), 119.0487 (63.6)3.34−8.5101,2,3,4,5,6
13.caffeic acid aC9H8O4179.0339179.0339 (20.5), 135.0437 (100), 117.0332 (0.64), 107.0489 (1.4)3.55−5.9321,2,3,4,5,6
14.ferulic acid O-hexosideC16H20O9355.1036355.1036 (18.1), 295.0817 (2.1), 265.0713 (0.7), 235.0609 (3.6), 193.0497 (22.1), 175.0390 (100), 160.0154 (50.5), 149.0593 (1.1), 134.0593 (1.1), 132.0203 (12.1)3.710.3511,2,3,4,5,6
15.sinapic acid O-dihexosideC23H32O15547.1669547.1669 (58.7), 487.1464 (1.3), 223.0607 (100), 205.0499 (71.9), 190.0263 (62.3), 175.0026 (34.1), 149.0230 (23.4), 119.0124 (6.3)3.800.1581,3,4,5,6
16.sinapic acid O-hexosideC17H22O10385.1143385.1143 (29.6), 325.0927 (1.8), 295.0809 (0.7), 265.0717 (3.5), 223.0607 (22.9), 205.0499 (100), 190.0262 (40.1), 175.0026 (27.7), 149.0232 (4.2), 119.0124 (4.2)3.820.7011,2,3,4,5,6
17.vanillic acid O-hexosideC14H18O9329.0888329.1144 (100), 285.1242 (43.7), 167.0603 (82.7)4.032.9621,2,3,4,5,6
18.o-coumaric acid aC9H8O3163.0389163.0389 (9.9), 135.0438 (1.3), 119.0487 (100)4.56−7.0991,2,3,4,5,6
19.ferulic acid aC10H10O4193.0497193.0497 (100), 178.0259 (3.5), 161.0232 (20.1), 134.0361 (7.6), 133.0281 (12.5), 6.51−4.8251,2,3,4,5,6
Flavonoids
20.luteolin 6,8-C-hexosideC27H30O16609.1468609.1468 (100), 519.1131 (4.7), 489.1039 (16.4), 471.0955 (2.4), 429.0829 (7.5), 399.0723 (27.4), 369.0618 (29.4), 339.0516 (3.5), 311.0564 (4.6), 175.0392 (1.74), 133.0280 (6.01)3.611.1201,2,4,5
21.luteolin C,O-dihexosideC27H30O16609.1468609.1468 (100), 489.1036 (1.9), 447.0934 (22.9), 369.0618 (1.8), 357.0615 (13.6), 327.0511 (49.2), 313.0354 (13.3), 285.0401 (3.9), 299.0558 (10.1), 298.0408 (5.7), 175.0392 (1.5), 133.0280 (4.9)3.851.1201,2,4,5,6
22.apigenin 6,8-diC-hexosideC27H30O15593.1517593.1517 (100), 503.1192 (3.4), 473.1095 (15.6), 413.0893 (0.9), 395.0784 (2.4), 383.0778 (19.8), 353.0668 (30.8), 325.0719 (1.2), 297.0766 (11.9), 161.0232 (1.2), 117.0331 (3.4),4.010.6011,2,3,4,5,6
23.luteolin C,O-hexoside isomerC27H30O16609.1466609.1466 (100), 489.1053 (1.1), 447.0929 (13.4), 369.0617 (1.5), 357.0616 (23.8), 327.0511 (78.8), 313.0352 (9.3), 285.0400 (3.8), 299.0565 (11.2), 298.0487 (7.8), 175.0387 (1.8), 133.0282 (8.8)4.070.8241,2,4,5,6
24.luteolin 6-C-hexoside-8-C-pentosideC26H28O15579.1362579.1362 (100), 561.1286 (0.9), 519.1138 (2.7), 489.1041 (16.6), 429.0825 (7.9), 411.0732 (2.9), 399.0724 (29.9), 381.0612 (1.9), 369.0617 (28.8), 339.0505(4.2), 311.0563 (3.9), 299.0557 (1.7), 298.0483 (4.44), 175.0390 (1.6), 133.0281 (6.0)4.101.2891,2,3,4,5,6
25.saponarin aC27H30O15593.1512593.1517 (100), 473.1094 (4.4), 431.0982 (8.4), 341.0666 (3.8), 311.0563 (28.2), 297.0403 (19.3), 269.0458 (3.5), 225.0546 (0.4), 161.0237 (1.9), 117.0332 (3.5)4.370.8041,2,3,4,5,6
26.apigenin 6-C-hexoside-8-C-pentosideC26H28O14563.1406563.1411 (100), 503.1190 (0.8), 473.1087 (11.4), 443.0981 (14.7), 425.0881 (0.8), 413.0887 (2.5), 383.0779 (12.9), 365.0659 (1.5), 353.0669 (20.7), 325.0717 (2.1), 323.0574 (1.7), 297.0769 (10.2), 283.0611 (2.6), 175.0388 (0.8), 135.0434 (1.8), 117.0331 (3.7)4.500.7481,2,3,4,5,6
27.homoorientin aC21H20O11447.0936447.0936 (100), 369.0632 (2.2), 357.0618 (37.3), 339.0519 (3.7), 327.0511 (59.3), 311.0558 (3.1), 299.0561 (9.5), 298.0470 (37.3), 297.0406 (12.6), 285.0407 (7.7), 133.0282 (12.8), 175.0393 (2.5)4.530.4371,2,3,4,5,6
28.chrysoeriol C-O-dihexosideC28H32O16623.1625623.1625 (100), 503.1204 (4.5), 461.1087 (4.2), 371.0773 (2.2), 341.0665 (31.4), 327.0514 (16.2), 312.0276 (11.6), 299.0542 (1.9), 298.0408 (8.9), 340.0584 (0.5), 298.0480 (8.9), 284.0330 (5.2), 269.0457 (14.3), 163.0025 (0.5), 178.9963 (0.43)4.601.2551,2,3,4,5,6
29.luteolin 7-O-sophoroside-6-C-hexosideC36H36O19771.1778771.1786 (100), 447.0932 (34.8), 429.0760 (1.1), 369.0597 (1.1), 357.0617 (27.0), 339.0501 (4.3), 327.0510 (80.4), 299.0560 (29.3), 285.0400 (4.4), 269.0493 (0.9), 297.0408 (8.9), 133.0280 (17.6)4.611.0222
30.orientin aC21H20O11447.0937447.0932 (95.5), 369.0605 (3.0), 357.0616 (32.5), 339.0504 (2.8), 327.0509 (100), 311.0558 (0.7), 299.0560 (10.0), 298.0479 (6.8), 297.0402 (12.8), 285.0399 (4.9), 165.0181 (2.1), 133.0281 (17.7), 107.0123 (0.4)4.68−0.2561,2,3,4,5,6
31.apigenin 6-C-hexoside-8-C-pentoside isomerC26H28O14563.1408563.1408 (100), 503.1183 (0.4), 473.1093 (12.2), 443.0884 (19.3), 413.0893 (2.1), 383.0776 (13.7), 353.0667 (23.4), 325.0721 (2.2), 311.0569 (1.8), 297.0764 (10.4), 283.0612 (2.6), 282.0531 (1.5), 281.0451 (0.6), 175.0387 (1.0), 135.044 (1.4), 117.0331 (4.1)4.760.3222,4,5,6
32.apigenin 2″-O-hexosyl-6-C-hexosideC27H30O15593.1512593.1516 (100), 413.0876 (3.8), 395.0770 (4.6), 377.0675 (3.0), 353.0670 (21.6), 325.0708 (2.4), 311.0563 (74.3), 283.0612 (22.7), 293.0469 (3.2), 282.0536 (1.5), 281.0451 (3.8), 269.0457 (0.3), 175.0024 (0.6), 117.0330 (5.5)4.880.6011,2,3,4,5,6
33.apigenin O-pentosyl-6-C-hexosideC26H28O14563.1406563.1411 (100), 443.0986 (08), 383.0772 (2.0), 353.0669 (4.3), 341.0665 (28.1), 323.0561 (2.7), 311.0562 (89.7), 283.0611 (29.5), 282.0554 (1.2), 281.0454 (5.5), 269.0451 (2.0), 237.0553 (0.6), 121.0280 (0.4), 117.0331 (6.6)4.990.7481,2,3,5,6
34.luteolin O-synapoylhexosyl-6-C-hexosideC38H40O20815.2040815.2053 (100), 447.0936 (21.0), 357.0616 (18.5), 341.0665 (24.3), 327.0512 (79.7), 313.0356 (11.1), 299.0558 (17.1), 298.0477 (15.9), 297.0406 (10.4), 285.0399 (4.1), 133.0279 (11.1)5.021.5741,2,3,4,5,6
35.rutin aC27H30O16609.1467609.1467 (100), 301.0349 (36.2), 300.0275 (67.0), 271.0247 (35.0), 255.0294 (14.4), 227.0343 (2.5), 211.0391 (1.4), 199.0388 (0.4), 178.9975 (2.4), 163.0021 (0.6), 151.0023 (5.7), 135.0069 (0.6), 107.0123 (2.2)5.070.9232,3,4,5,6
36.apigenin O-caffeoylhexosyl-8-C-hexosideC36H36O18755.1829755.1838 (92.7), 431.0985 (45.1), 413.0886 (1.6), 353.0638 (2.1), 341.0667 (27.1), 323.0538 (2.7), 311.0564 (100), 283.0612 (40.1), 281.0453 (4.5), 269.0436 (1.4), 161.0231 (15.2), 133.0278 (8.9), 117.0391 (7.8)5.131.2621,2,4,5
37.isovitexin aC21H20O10431.0985431.0985 (100), 412.0894 (1.4), 341.0669 (18.7), 323.0570 (4.1), 311.0565 (58.4), 283.0614 (18.2), 269.0452 (2.9), 117.0330 (7.7), 161.0233 (2.4), 135.0441 (1.7)5.140.3941,2,3,4,5,6
38.luteolin O-feruloylhexosyl-8-C-hexosideC37H38O19785.1935785.1940 (100), 447.0940 (28.4), 369.0600 (2.1), 357.0618 (24.0), 339.0502 (1.9), 327.0510 (92.8), 299.0566 (23.2), 298.0485 (14.1), 297.0408 (10.1), 341.0658 (18.4), 285.0413 (2.6), 175.0392 (5.0), 133.0282 (13.0)5.200.7361,2,4,5
39.luteolin 7-O-coumaroylhexosyl-6-C-hexosideC36H36O18755.1829755.1837 (100), 447.0936 (30.7), 369.0602 (2.2), 357.0618 (23.3), 339.0506 (2.7), 327.0511 (76.6), 299.0562 (18.4), 298.0480 (6.6), 297.0401 (5.2), 285.0393 (2.9), 163.0387 (2.2), 145.0278 (3.2), 133.0279 (8.7), 119.0488 (0.6), 117.0331 (2.5), 5.271.1031,2,4,5
40.eryodictiol 7-O-hexosideC21H22O11449.1089449.1094 (14.8), 287.0561 (100), 151.0023 (61.8), 135.0437 (50.5), 125.0228 (4.1), 107.0123 (13.0)5.271.0581,2,3,4,5,6
41.chrysoeriol 7-O-caffeoylhexosyl-8-C-hexosideC37H38O19785.1935785.1936 (91.8), 461.1106 (36.0), 371.0775 (29.5), 341.0666 (100), 327.0528 (12.7), 298.0479 (60.7), 299.0531 (6.9), 297.0406 (5.5), 161.0232 (13.3), 133.0283 (11.3)5.280.1891,2,4,5
42.isoquercitrin aC21H20O12463.0882463.0885 (100), 301.0349 (44.1), 300.0276 (75.1), 271.0248 (37.1), 255.0296 (0.7), 243.0299 (9.3), 227.0342 (2.4), 211.0391 (0.6), 199.0392 (0.5), 178.9969 (3.0), 163.0027 (1.9), 151.0024 (5.3), 135.0072 (0.5), 121.0280 (1.6), 107.0123 (2.8)5.280.7141,2,3,4,5,6
43.luteolin-7-O-glucoside aC21H20O11447.0937447.0937 (100), 285.0404 (86.7), 284.0327 (36.3), 255.0302 (1.2), 227.0346 (1.9), 211.0397 (1.1), 151.0024 (4.7), 133.0282 (4.2), 107.0124 (3.3)5.370.8401,2,3,4,5,6
44.chrysoeriol 6-C-hexosideC22H22O11461.1089461.1093 (100), 371.0422 (21.2), 353.0674 (2.8), 341.0665 (69.0), 313.0727 (3.2), 312.0609 (0.5), 299.0520 (4.8), 298.0481 (14.1), 297.0404 (13.3), 284.0327 (1.7), 269.0455 (2.9), 255.0294 (1.4), 133.0284 (2.2)5.430.8141,2,3,4,5,6
45.apigenin 7-O-synapoylhexosyl-8-C-hexosideC38H40O19799.2103799.2103 (88.3), 431.0984 (53.7), 341.0665 (29.8), 323.0559 (3.8), 311.0563 (100), 283.0610 (44.5), 282.0533 (9.9), 281.0463 (7.4), 190.0265 (4.8), 175.0025 (1.8)5.461.5371,2,4,5
46.chrysoeriol 7-O-dihexosideC28H32O16623.1620623.1620 (27.1), 299.0561 (100), 284.0326 (40.6), 255.0289 (4.1), 151.0026 (0.9)5.490.4694, 5
47.kaempferol 3-O-rutinoside aC27H30O15593.1512593.1518 (100), 285.0403 (82.5), 284.0327 (47.6), 255.0298 (37.7), 227.0345 (24.2), 211.0397 (2.5), 135.0071 (1.5), 151.0024 (1.8), 107.0124 (2.4)5.631.0061,2,4,5,6
48.apigenin O-feruloylhexosyl-8-C-hexosideC38H38O18769.200769.1996 (83.5), 431.0985 (52.3), 353.0680 (3.5), 341.0666 (23.9), 323.0564 (5.2), 311.0563 (100), 283.0611 (41.8), 282.0521 (4.6), 281.0452 (6.7), 269.0443 (2.0), 175.0391 (2.1), 117.0332 (9.1)5.701.3821,2,3,4,5,6
49.chrysoeriol O-feruloylhexoside-8-C-hexosideC38H40O19799.2091799.2111 (60.9), 461.1094 (83.2), 371.0762 (31.7), 353.0687 (2.1), 341.0667 (100), 309.0394 (7.2), 299.0500 (2.9), 298.0483 (54.9), 297.0399 (13.5), 175.0387 (2.8), 160.0146 (1.7), 132.0201 (2.4)5.782.5251,2,4,5
50.isorhamnetin 3-O-rutinoside aC28H32O18623.1618623.1622 (100), 315.0509 (95.2), 300.0269 (13.6), 299.0201 (15.1), 271.0248 (33.7), 255.0295 (12.3), 243.0297 (15.8), 227.0345 (4.3), 1561.0022 (1.6)5.780.7581,3,4,5,6
51.apigenin O-coumaroylhexosyl-8-C-hexosideC36H36O17739.1880739.1893 (87.0), 431.0983 (39.6), 413.0881 (1.1), 253.0668 (2.6), 341.0667 (25.1), 323.0562 (5.9), 311.0564 (100), 283.0612 (44.0), 282.0534 (2.4), 281.0468 (4.4), 145.0282 (5.5), 117.0331 (10.9)5.801.8361, 2, 3, 4, 6
52.kaempferol 3-O-glucoside aC21H20O11447.0934447.0935(100), 285.0398 (21.9), 284.0327 (52.1), 255.0297 (39.5), 227.0346 (39.1), 211.0394 (0.7), 178.9966 (0.7), 151.0027 (1.8), 107.0126 (0.6)5.850.4821,2,3,4,5,6
53.chrysoeriol O-coumaroylhexoside-8-C-hexosideC37H38O18769.2000769.2000 (83.7), 461.1091 (62.3), 371.0771 (30.1), 341.0667 (100), 353.0651 (1.5), 309.0401 (6.6), 298.0481 (67.2), 299.0519 (6.7), 297.0400 (14.6), 284.0330 (2.1),5.921.8371,2,4,5
54.apigenin 7-O-neohesperidosideC27H30O14577.1568577.1568 (69.4), 431.0989 (0.5), 413.0882 (1.0), 269.0454 (100), 211.0390 (0.8), 151.0023 (2.2), 149.0230 (0.8), 117.0330 (3.5), 107.0121 (1.8)6.000.9031,2,3,4,5,6
55.apigenin 7-O-glucoside aC21H20O10431.0985431.0985 (100), 269.0449 (26.9), 268.0378 (61.1), 211.0394 (1.8), 151.0025 (2.9), 149.0228 (1.2), 107.0124 (2.5), 117.0331 (2.1)6.040.3251,2,3,4,5,6
56.chrysoeriol 7-O-rutinosideC28H32O15607.1668607.1676 (89.6), 299.0561 (100), 284.0327 (43.3), 255.0299 (43.3)6.201.2461,2,3,4,5,6
57.chrysoeriol 7-O-hexosideC22H22O11461.1081461.1081 (100), 446.0857 (24.6), 371.0756 (0.2), 341.0648 (0.6), 300.0590 (0.9), 299.0555 (9.3), 298.0482 (12.6), 283.0248 (18.1), 255.0297 (68.6), 227.0348 (0.3), 211.0398 (0.2), 151.0027 (0.7), 117.0331 (1.7), 107.0124 (0.1)6.25−1.8321,2,3,4,5,6
58.cirsiliol O-hexosideC23H24O12491.1195491.1191 (100), 476.0944 (21.6), 461.0748 (5.1), 329.0676 (3.7), 328.0594 (6.6), 314.0442 (3.8), 313.0351 (6.4), 299.0198 (6.1), 285.0397 (12.4), 243.0292 (10.3)6.30−0.8541,2,4,5,6
59.luteolin 2″-O-coumaroyl-6-C-hexosideC30H26O13593.1301593.1309 (100), 473.0900 (2.3), 447.0922 (31.5), 429.0841 (13.5), 357.0603 (13.6), 339.0526 (2.1), 327.0511 (38.8), 309.0406 (46.4), 299.0560 (12.9), 297.0399 (2.0), 285.0432 (1.4)6.711.3421,2,3,6
60.luteolin O-coumaroyldeoxyhexosyl-6-hexoside C36H36O17739.1880739.1893 (100), 447.0944 (12.3), 369.0611 (1.2), 357.0617 (13.8), 339.0518 (1.7), 327.0509 (58.8), 313.0355 (20.1), 299.0552 (13.8), 298.0485 (10.1), 297.0402 (7.0), 285.0396 (5.9), 269.0457 (1.1), 163.0396 (1.3), 151.0024 (0.8), 133.0281 (8.9)6.751.8361,2,3,4,5
61.apigenin 2″-O-coumaroyl-6-C-hexosideC30H26O12577.1351577.1365 (7.4), 431.0986 (100), 413.0882 (77.5), 341.0669 (21.3), 323.0593 (3.0), 311.0505 (65.4), 293.0457 (96.2), 283.1061 (35.7), 269.0435 (1.6), 175.0026 (15.2), 163.0389 (20.3), 119.0487 (34.1), 117.0332 (12.7)7.422.4101,2,5,6
62.apigenin O-coumaroyldeoxyhexosyl-6-C-hexosideC36H36O16723.1931723.1941 (100), 431.0979 (8.5), 341.0677 (5.4), 311.0564 (53.5), 297.0403 (27.2), 283.0611 (20.3), 282.0534 (9.6), 281.0454 (6.0), 269.0457 (4.9), 163.0387 (2.1), 135.0436 (1.0), 117.0330 (8.1)7.471.4272,4,5
63.apigenin aC15H10O5269.0455269.0455 (100), 227.0363 (1.3), 151.0027 (6.5), 149.0229 (4.4), 117.0331 (18.6), 107.0123 (4.5),8.62−0.9171,2,3,4,5,6
64.luteolin aC15H10O6285.0404285.0404 (100), 241.0493 (0.4), 151.0025 (4.1), 149.0233 (1.9), 133.0280 (20.4), 107.0124 (3.6)8.91−0.0711,2,3,4,5,6
65.chrysoeriolC16H12O6299.0561299.0561 (100), 284.0327 (81.0), 256.0375 (16.6), 227.0346 (2.9), 151.0025 (3.1), 107.0123 (2.2)8.91−0.0711,2,3,4,5,6
Amino acids and derivatives
66.N-hexosylglutamic acidC11H17O8N290.0883290.0883 (8.82), 272.0277 (2.2), 254.0680 (1.3), 230.0668 (2.9), 200.0556 (44.1), 170.0448 (8.8), 128.0338 (100), 84.0438 (1.7)0.722.6151,2,3,4,5,6
67.N-hexosylvalineC11H21O7N278.1246278.1246 (1.1), 260.1139 (1.1), 188.0918 (6.2), 158.0810 (4.3), 146.0442 (0.5), 116.0701 (100), 101.0228 (8.8), 85.0280 (0.3)0.900.1611,3,4,5,6
68.N-hexosyltyrosineC15H21O8N342.1197342.1171 (3.8), 324.1084 (0.6), 282.0967 (0.5), 252.0877 (11.5), 222.0766 (9.1), 180.0656 (100), 163.0388 (11.1), 119.0487 (10.2)0.920.6441,2,3,4,5,6
69.N-hexosylleucineC15H23O7N292.1400292.1400 (1.3), 274.1300 (0.4), 232.1179 (0.3), 202.1078 (7.9), 172.0968 (3.1), 152.1058 (0.3), 130.0859 (100)1.071.1061,2,3,4,5,6
70.N-hexosylphenylalanineC15H21O7N326.1248326.1248 (1.8), 308.1145 (1.1), 266.1046 (0.6), 236.0925 (11.2), 206.0816 (6.1), 186.0916 (0.4), 164.0705 (100), 147.0438 (18.9), 119.0485 (1.8), 101.0229 (14.2)1.530.7201,2,3,4,5,6
71.phenylalanineC9H11O2N164.0705164.0705 (53.3), 147.0438 (100), 146.0594 (0.5), 135.0437 (13.0), 119.0489 (8.9), 103.9187 (2.2)1.54−7.5081,2,3,4,5,6
72.N-hexosyltryptophanC17H22O7N2365.1360365.1357 (0.6), 275.1040 (10.5), 245.0924 (3.3), 203.0801 (100), 142.0647 (5.9), 159.0915 (2.3)2.361.7141,2,3,4,5,6
73.tryptophanC11H12O2N2203.0818203.0818 (71.0), 186.0548 (4.4), 159.0916 (19.9), 142.0649 (25.7), 116.0491 (100), 74.0231 (31.2), 72.0075 (30.9)2.482.7661,2,3,4,5,6
74.γ-glutamyl-leucineC11H20O5N2259.1300241.1191 (20.0), 223.1083 (10.5), 215.1400 (0.4), 197.1287 (11.1), 130.0859 (64.9), 128.0339 (100), 127.0499 (0.8)2.820.2121,2,3,4,5,6
Fatty acids
75.trihydroxyoctadecadienoic acidC18H32O5327.2177327.2177 (100), 309.2070 (0.8), 291.1971 (3.90), 247.2048 (0.5), 239.1286 (3.1), 229.1440 (3.9), 211.1331 (20.4), 183.1382 (2.2), 171.1014 (6.4), 155.1071 (0.6), 127.1112 (0.7), 97.0643 (3.3), 85.0277 (3.9), 70.1990 (0.9)9.130.1301,2,3,4,5,6
76.trihydroxyotadecenoic acidC18H34O5329.2334329.2334 (100), 311.2224 (1.0), 293.2121 (0.9), 229.1439 (18.8), 211.1332 (24.6), 199.1344 (0.3), 183.1379 (2.9), 171.1014 (4.1), 155.1066 (0.3), 139.111 (1.42), 127.1111 (1.1), 99.0799 (3.6), 83.0488 (0.3), 70.1988 (0.3)9.780.0691,2,3,4,5,6
77.trihydroxyoctadecatrienoic acidC18H30O5325.2019325.2019 (100), 307.1915 (19.7), 289.1807 (1.9), 263.2015 (12.5), 245.1904 (0.4), 235.2053 (0.9), 209.0526 (0.2), 169.1218 (0.4), 137.0956 (3.9), 125.0957 (2.9), 97.0642 (0.3), 83.0484 (0.9), 57.0328 (1.3)11.85−0.4531,2,3,4,5,6
78.dihydroxyoctadecadienoic acidC18H32O4311.2229311.2229 (100), 293.2127 (6.6), 275.2008 (4.0), 235.1698 (4.16), 223.1697 (39.9), 208.2670 (0.3), 196.1057 (1.5), 113.0954 (1.1), 87.0435 (12.4), 57.0330 (5.3)12.510.3771,2,3,4,5,6
79.dihydroxyoctadecenoic acidC18H34O4313.2385313.2385 (100), 295.2277 (9.5), 277.2174 (2.6), 209.1976 (0.2), 195.1382 (2.7), 183.1380 (25.5), 129.0907 (16.5), 99.0799 (10.5)13.460.1831,2,3,4,5,6
80.dihydroxyoctadecenoic acidC18H34O4313.2385313.2385 (100), 295.2277 (5.5), 277.2174 (4.6), 201.1124 (43.2), 171.1014 (5.9), 155.1065 (1.0), 139.1116 (0.7), 127.1113 (3.9) 13.720.1831,2,3,4,5,6
81.dihydroxyoctadecatrienoic acidC18H30O4309.2071309.2071 (100), 291.1966 (28.6), 273.1864 (5.00), 263.2047 (0.4), 247.2063 (26.6), 139.1116 (0.6), 70.2070 (0.8), 57.0329 (1.9)14.61−0.1381,2,3,4,5,6
82.dihydroxyoctadecanoic acidC18H36O4315.2540315.2540 (100), 297.2435 (5.6), 201.1130 (0.3), 171.1015 (0.9), 155.1064 (0.2), 141.1269 (2.3), 127.1113 (1.1)14.85−0.3851,2,3,4,5,6
83.linoleic acidC18H32O2279.2327279.2327 (100), 136.1251 (0.4), 70.0576 (0.7)21.96−1.053,6
a—compare to reference standard; 1—E. intortum leaves; 2—E. intortum flowers; 3—E. intortum tuber; 4—E. spiculatum leaves; 5—E. spiculatum flowers; 6—E. spiculatum tuber.
Table
Table 3. Antioxidant properties of the tested extracts.
Table 3. Antioxidant properties of the tested extracts.
SpeciesPartsDPPH
(mg TE/g)		ABTS
(mg TE/g)		CUPRAC
(mg TE/g)		FRAP
(mg TE/g)		PBD
(mmol TE/g)		MCA
(mg EDTAE/g)
Eminium intortumLeaves32.20 ± 1.26 a52.10 ± 0.83 a88.27 ± 1.49 a33.13 ± 0.68 a0.96 ± 0.01 b63.43 ± 0.70 a
Flowers27.87 ± 0.75 b40.44 ± 1.20 c55.29 ± 1.78 c24.67 ± 1.22 b1.38 ± 0.08 a36.05 ± 1.22 b
Tubers26.90 ± 1.61 b46.80 ± 0.86 b71.51 ± 1.93 b32.58 ± 4.02 a1.48 ± 0.15 a39.15 ± 2.79 b
Eminium spiculatumLeaves26.58 ± 1.00 b54.34 ± 0.53 a86.27 ± 2.74 a29.90 ± 0.75 b1.08 ± 0.03 b61.55 ± 3.97 a
Flowers20.73 ± 1.51 c48.45 ± 1.96 b70.06 ± 3.03 b30.92 ± 0.21 b1.23 ± 0.05 a30.92 ± 0.48 c
Tubers33.67 ± 1.26 a51.86 ± 2.14 ab75.43 ± 4.47 b41.33 ± 1.25 a1.02 ± 0.07 b53.00 ± 1.88 b
Values are reported as mean ± SD of three parallel measurements. TE: Trolox equivalent; PBD: Phosphomolybdenum assay; MCA: Metal chelating assay; EDTAE: EDTA equivalent. Different letters indicate significant differences between the tested extracts (p < 0.05).
Table
Table 4. Enzyme inhibitory effect of the tested extracts.
Table 4. Enzyme inhibitory effect of the tested extracts.
SpeciesPartsAChE
(mg GALAE/g)		BChE
(mg GALAE/g)		Tyrosinase
(mg KAE/g)		Amylase (mmol ACAE/g)Glucosidase (mmol ACAE/g)
Eminium intortumLeaves1.27 ± 0.09 cna35.37 ± 0.74 c0.30 ± 0.01 b0.35 ± 0.02 c
Flowers2.25 ± 0.07 b0.67 ± 0.08 b40.66 ± 0.61 b0.31 ± 0.01 ana
Tubers2.64 ± 0.01 a2.01 ± 0.07 a48.13 ± 0.24 a0.26 ± 0.01 cna
Eminium spiculatumLeaves2.64 ± 0.10 b1.11 ± 0.20 b42.58 ± 1.28 b0.27 ± 0.01 a0.99 ± 0.02 a
Flowers2.72 ± 0.03 a1.89 ± 0.07 a37.74 ± 1.94 c0.24 ± 0.01 b0.89 ± 0.01 b
Tubers2.66 ± 0.05 a1.69 ± 0.13 a50.73 ± 2.29 a0.24 ± 0.01 bna
Values are reported as mean ± SD of three parallel measurements. GALAE: Galantamine equivalent; KAE: Kojic acid equivalent; ACAE: Acarbose equivalent; na: not active. Different letters indicate significant differences between the tested extracts (p < 0.05).
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MDPI and ACS Style

Babacan, E.Y.; Zheleva-Dimitrova, D.; Gevrenova, R.; Bouyahya, A.; Balos, M.M.; Cakilcioglu, U.; Sinan, K.I.; Zengin, G. Orbitrap Mass Spectrometry-Based Profiling of Secondary Metabolites in Two Unexplored Eminium Species and Bioactivity Potential. Plants 2023, 12, 2252. https://doi.org/10.3390/plants12122252

AMA Style

Babacan EY, Zheleva-Dimitrova D, Gevrenova R, Bouyahya A, Balos MM, Cakilcioglu U, Sinan KI, Zengin G. Orbitrap Mass Spectrometry-Based Profiling of Secondary Metabolites in Two Unexplored Eminium Species and Bioactivity Potential. Plants. 2023; 12(12):2252. https://doi.org/10.3390/plants12122252

Chicago/Turabian Style

Babacan, Ebru Yuce, Dimitrina Zheleva-Dimitrova, Reneta Gevrenova, Abdelhakim Bouyahya, Mehmet Maruf Balos, Ugur Cakilcioglu, Kouadio Ibrahime Sinan, and Gokhan Zengin. 2023. "Orbitrap Mass Spectrometry-Based Profiling of Secondary Metabolites in Two Unexplored Eminium Species and Bioactivity Potential" Plants 12, no. 12: 2252. https://doi.org/10.3390/plants12122252

APA Style

Babacan, E. Y., Zheleva-Dimitrova, D., Gevrenova, R., Bouyahya, A., Balos, M. M., Cakilcioglu, U., Sinan, K. I., & Zengin, G. (2023). Orbitrap Mass Spectrometry-Based Profiling of Secondary Metabolites in Two Unexplored Eminium Species and Bioactivity Potential. Plants, 12(12), 2252. https://doi.org/10.3390/plants12122252

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