A Comprehensive Phytochemical Analysis of Sideritis scardica Infusion Using Orbitrap UHPLC-HRMS

Dimitrina Zheleva-Dimitrova; Yulian Voynikov; Reneta Gevrenova; Vessela Balabanova

doi:10.3390/molecules29010204

Abstract

Sideritis scardica Griseb, also known as “mountain tea” and “Olympus tea” (Lamiaceae family) is an endemic plant from the mountainous regions of the Balkan Peninsula. In this study, we focused on an in-depth phytochemical analysis of S. scardica infusion using ultra-high-performance liquid chromatography hyphenated with high-resolution mass spectrometry (UHPLC–HRMS). Quantitative determination of the main secondary metabolites was carried out by UHPLC–HRMS analyses using the external standard method. The results revealed more than 100 metabolites, including five sugar acids and saccharides, 21 carboxylic, hydroxybenzoic, hydroxycinnamic acids, and derivatives, 15 acylquinic acids, 10 phenylpropanoid glycosides, four iridoid glycosides, 28 flavonoids, seven fatty acids, and four organosulfur compounds. Furthermore, a dereplication and fragmentation patterns of five caffeic acids oligomers and four acylhexaric acids was performed for the first time in S. scardica. Regarding the quantitative analysis, the phenylethanoid verbascoside (53) (151.54 ± 10.86 mg/g lyophilized infusion, li), the glycosides of isoscutellarein (78) (151.70 ± 14.78 mg/g li), methylisoscutelarein (82) (107.4 ± 9.07 mg/g li), and hypolaetin (79) (78.33 ± 3.29 mg/g li), as well as caffeic acid (20) (87.25 ± 6.54 mg/g li), were found to be the major compounds in S. scardica infusion. The performed state-of-the-art phytochemical analysis of S. scardica provides additional knowledge for the chemical constituents and usage of this valuable medicinal plant.

Keywords:

Sideritis scardica Griseb; UHPLC–HRMS; phytochemical analysis

1. Introduction

Sideritis scardica Griseb (Lamiaceae family) is an endemic plant of the mountainous regions of the Balkan Peninsula [1,2]. It is often referred to as “mountain tea”, “ironwort”, “Olympus tea”, and “Pirin tea” [3]. Mountain tea is a perennial herbaceous plant with a well-developed root system, the stem is 15–40 cm and woody at the base, the leaves are opposite with gray hairs, the flowers are clustered in a dense spike, the middle bracts are 12–20 mm long, i.e., longer than the flowers, the corolla is lemon yellow with glandules, and the calyx is tubular-campanulate [2,3]. Usually, Sideritis plants are applied in traditional medicine, mostly as an aromatic herbal tea [4,5,6,7]. The tea is made from the aerial parts of the plant by infusion or decoction [8]. Historically, S. scardica has been used to treat inflammation, common colds, asthma, bronchitis, and gastrointestinal disorders. It is supposed to relieve pain, including rheumatic pain, as well as reducing stress and anxiety. The plant name comes from the Greek word “sideros”, meaning “iron”, as it was used in ancient times to heal wounds from iron weapons [4]. Regular consumption of mountain tea by rats has been shown to lead to weight loss and prevent insulin resistance by lowering blood glucose and triglyceride levels and increasing liver glycogen content [8]. Additionally, antioxidant properties and positive effects on memory and cognitive abilities have also been observed [7,9,10]. Sideritis species have also been used topically on the skin and as an antiseptic solution to sooth the pain of tooth extraction [3].

The traditional medicinal usage of the species is based on the phytochemical constituents, including phenolic acids (chlorogenic acid, 3-caffeoylquinic acid, feruloylquinic acid, and others), flavonoids and their derivatives (hypolaetin, isoscutellarein, and others), phenylethanoid glycosides (lavandulifolioside, verbascoside, echinacoside, allysonoside, and others), and terpenoids (mostly iridoid glycosides) [6,8,11,12]. These chemical compounds have been explored in phytochemical studies, operating with various extraction techniques such as hydrodistillation and solvent and supercritical extractions [8,12,13,14,15]. Precisely, the most abundant secondary metabolites of S. scardica water extracts (i.e., when making infusion or decoction) are flavonoids, hydoxycinnamic acid derivatives, and phenylethanoid glycosides [7,14,16,17]. Identification of closely related species of the genus Sideritis is based on the dominant 5-caffeoylquinic acid, lavandulifolioside, verbascoside, isoscutellarein 7-O-allosyl(1→2)glucoside, hypolaetin 7-O-[6‴O-acetyl]-allosyl(1→2)glucoside, isoscutellarein 7-O-[6‴-O-acetyl]-allosyl(1→2) glucoside, 3′-O-methylhypolaetin 7-O-[6‴-O-acetyl]-allosyl(1→2)glucoside, and 4′-O-methylhypolaetin 7-O-[6‴-O-acetyl]-allosyl-(1→2)-[6″-O-acetyl]-glucoside. Thus, these compounds have been used as chemotaxonomical markers [6].

Based on the literature available on Sideritis scardica, there is no detailed metabolite profiling of the species, something which seems important in light of its health benefits. An in-depth UHPLC–HRMS analysis of the main metabolites of S. scardica, together with quantitative determination, was conducted. More than 100 secondary metabolites were identified/tentatively elucidated in a lyophilized infusion of mountain tea. The performed phytochemical analysis of S. scardica will provide additional knowledge of the chemical constituents and usage of this valuable medicinal plant for the future.

2. Results and Discussion

2.1. Metabolite Profiling of S. scardica Lyophilized Infusion

Herein, an in-depth UHPLC–HRMS analysis of S. scardica infusion was conducted by allowing the dereplication/annotation of 103 metabolites, including five sugar acids and saccharides, 21 carboxylic, hydroxybenzoic, hydroxycinnamic acids, and derivatives, five caffeic acids oligomers, 15 acylquinic acids, four acylhexaric acids, 10 phenylpropanoid glycosides, four iridoid glycosides, 28 flavonoids, seven fatty acids, and four organosulfur compounds (Table 1). This study allowed the identification of caffeic acids oligomers, acylquinic, acylhexaric acids, and flavonoids not previously reported in the taxon. The total ion chromatogram (TIC) in negative ion mode of the studied extract is presented in Figure 1.

Figure 1. Total ion chromatogram (TIC) in negative ion mode of Sideritis scardica extract; the same chromatogram 2–7 min. For compound numbering see Table 1.

2.1.1. Sugar Acids and Saccharides

Compound 1 ([M−H]⁻ at m/z 165.041) gave fragment ions at m/z 147.02 [M−H−H₂O]⁻, 129.018 [M−H−2H₂O]⁻, 111.01 [M−H−3H₂O]⁻, and 101.023 [M−H−3H₂O−CO]⁻, as well as ions corresponding to the loss of 60 Da [M−H−C₂H₄O₂]⁻ and 90 Da [M−H−C₃H₆O₃]⁻, respectively. Thus, compound 1 was annotated as xylonic acid. Analogously, 4 was related to pentose with a base peak at m/z 75.00 (C₂H₂O₃). Moreover, 2 (with an additional CH₂ group compared to 4) and 3 (with additional CH₂O) were identified as hexose and gluconic acid, respectively (Table 1). The identity of asystoside (5) was suggested by the transitions 583.261→421.209→289.166→161.445, resulting from the losses of hexosyl (162.053 Da), pentosyl (132.043 Da), and oct-1-en-3-ol units (C₈H₁₆O, 128.122 Da) (Table 1). All above-mentioned compounds were previously identified in the species [12].

2.1.2. Carboxylic, Hydroxybenzoic, Hydroxycinnamic Acids and Their Derivatives

Eight hydroxybenzoic acids (9, 12, 15, 16, 18, 19, 21, and 24), four hydroxycinnamic acids (17, 20, 25, and 26) and their glycosides (10, 11, 13, 14, 22, and 23), together with quinic (6), oxaloglutaric (7), and citric acid (8), were identified based on the comparison with reference standards and literature data in the assayed extract (Table 1) [12,17]. Compound 8 ([M−H]⁻ at m/z 191.018) showed fragment ions at m/z 173.008 [M−H−H₂O]⁻, 147.028 [M−H−CO₂]⁻, a base peak at m/z 111.007 [M−H−CO₂−2H₂O]⁻, and was related to citric acid [18]. A key step in the dereplication of phenolic acid glycosides was the neutral losses of 162.05, 132.04, and 308.11 Da, corresponding to hexose, pentose, and rutinose, respectively, together with the base peaks of the respective phenolic acid deprotonated molecule. Thus, pentosylhexosides of hydroxybenzoic acid (10) and vanillic acid (11), dihexoside of caffeic acid (14), and hexoside of coumaric acid (22) were annotated. Compounds 13 ([M−H]⁻ at m/z 487.146) and 23 ([M−H]⁻ at m/z 501.161) gave base peaks at m/z 179.034 [caffeic acid−H]⁻ and 193.050 [ferulic acid−H]⁻, corresponding to the loss of rutinose ([M−H−rutinose]⁻), and were identified as caffeic acid O-rutinoside and ferulic acid O-rutinoside, respectively (Table 1, Figure 2). Compounds 10, 11, 12, 14, 15, 16, 17, 18, 19, 22, 23, 24, and 25 are reported for the first time in S. scardica.

Figure 2. MS/MS spectrum of ferulic acid O-rutinoside (23).

2.1.3. Caffeic Acids Oligomers

Caffeic acid oligomers consist of ester-bonded monomers such as danshensu, caffeic acid, and others and are present in Lamiaceae species [19,20,21]. Based on the accurate masses, MS/MS data, and literature data, a dimer rosmarinic acid (29), two trimers (27 and 30), and two tetramers (28 and 31) were dereplicated in the studied S. scardica extract. The fragmentation pattern and retention time of rosmarinic acid (29) were compared with reference standard. Key points in the caffeic acid oligomers annotation were the indicative fragment ions derived from the cleavage of a and b ester bonds with loss of danshensu [M−H−198.05]⁻, danshensoyl [M−H−180.04]⁻, and caffeoyl residue [M−H−162.03]⁻, respectively [19]. Compound 30 ([M−H]⁻ at m/z 491.099) afforded a base peak at m/z 311.056, corresponding to the easier loss of danshensu, due to the dibenzooxepin structure, restraining the cleavage of the a bond. Based on a comparison with literature data, 30 was tentatively identified as isosalvianolic acid C [19] (Table 1, Figure 3). With respect to 27, an abundant fragment ion at m/z 493.114 [M−H−CO₂]⁻ and a base peak at m/z 339.059 [M−H−198.05]⁻ were indicative of the presence of CO₂ group attached to the benzofuran ring and danshensu residue. This allowed us to deduce the structure of lithospermic acid [19,20]. The fragmentation pathway of 28 included prominent ions at m/z 673.157 [M−H−CO₂]⁻, 537.105 [M−H−180.04]⁻, 519.095 [M−H−198.05]⁻, 493.115 [M−H−180.04−CO₂]⁻, 339.051 [M−H−2 × 198.05]⁻, and a base peak at m/z 321.041 [M−H−198.05−180.04−CO₂]⁻, indicating, consequently, losses of two danshensu residues and carboxyl groups. Moreover, diagnostic ions at m/z 537.093, corresponding to deprotonated lythospermic acid, as well as the lack of loss of caffeoyl residue, suggested a terminal danshensu residue linked to lithospermic acid. Thus, compound 28 was dereplicated as salvianolic acid B (Table 1, Figure 3). Similarly, 31 was related to didehydrosalvianolic acid B (Table 1, Figure 3) [19]. Compounds 27, 28, 30, and 31 are reported for the first time in S. scardica.

Figure 3. MS/MS spectrum of (A) salvianolic acid B (28) and (B) isosalvianolic acid C (30).

2.1.4. Acylquinic Acid

Overall, three caffeoylquinic (33, 36, and 38), four p-coumaroylquinic (35, 41, 42, and 44), two syringoylquinic (37 and 40), three feruloylquinic (39, 43, and 45), together with two hexosides (32 and 34), and a syringoyl–caffeoylquinic acid (46) were dereplicated or annotated in the studied extract (Table 1). The acylquinic acids (AQAs) annotation was based on the fragment ions and their relative abundances corresponding to each subclass AQAs [22,23,24]. Three isobars shared the same deprotonated molecule [M−H]⁻ at m/z 353.086. Compound 36 was identified as chlorogenic acid (5-caffeylquinic acid) due to the base peak at m/z 191.055 [quinic acid−H]⁻. The positional isomer neochlorogenic acid (3-caffeylquinic acid) (33) was apparent by the higher relative abundances of the fragment ions at m/z 179.033 (61.2%) and 135.043 (48.7%) than those of 36. Compounds 33 and 36 were unambiguously identified by comparison with reference standards. In the MS/MS spectrum of 38, 40, and 42, a base peak at m/z 173.044 [quinic acid−H−H₂O]⁻ was detected, indicating caffeoyl, syringoyl, and p-coumaroyl residues at position 4 of the quinic acid. Thus, 38, 40, and 42 were annotated as 4-caffeylquinic, 4-syringoyl, and 4-p-coumaroyl acids, respectively [24]. The compounds 3-p-coumaroylquinic acid (3-p-CoQA) (35) and 3-feruloylquinic acid (3-FQA) (39) were identified from the base peaks at m/z 163.039 [p-CoA−H]⁻ and 193.050 [FA-H]⁻ (Table 1). Compounds 37, 41, and 43 showed a base peak at m/z 191.055 and fragment ions at m/z 197.045 [syringic acid−H]⁻, 163.038 [p-CoA−H]⁻, and 193.050 [FA−H]⁻, respectively, and were identified as 5-syringoylquinic, 5-p-coumaroylquinic, and 5-feruloylquinic (FQA) acids [22,23,24] (Table 1).

With respect to compound 46, the base peak at 197.045 [syringic acid-H]⁻, together with a diagnostic fragment ion at m/z 335.077 [CQA−H−H₂O]⁻ indicated syringoyl–caffeoylquinic acid. Additionally, two hexosides of neochlorogenic (32) and chlorogenic acid (34) were also dereplicated (Table 1).

2.1.5. Acylhexaric Acids

Key steps in the acylhexaric acids annotation were the subsequent losses of one hydroxydihydrocaffeoyl (47, 48) and two (49, 50) hydroxydihydrocaffeoyl and syringoyl residues (Table 1, Figure 4). Thus, the base peak in the MS/MS spectra was consistent with [hexaric acid (HA)−H]⁻ at m/z 209.030 (C₆H₉O₈) supported by the series of indicative ions at m/z 191.019 [HA-H-H₂O]⁻, 147.029 [HA−H−H₂O−CO₂]⁻, 129.018 [HA−H−2H₂O−CO₂]⁻, 111.007 [HA−H−3H₂O−CO₂]⁻, and 85.028 [HA−H−2H₂O−2CO₂]⁻ (Table 1) [25]. Compounds 49 and 50 shared the same [M−H]⁻ at m/z 569.116. They formed the prominent fragment ions at m/z 389.073 [M−H−180.04]⁻, 371.063 [M−H−198.05]⁻, and 209.030 [M−2 × 180.04]⁻, resulting from the concomitant loss of hydroxydihydrocaffeoyl and syringoyl residues. Syringoyl moiety was suggested by the fragment ions at m/z 197.045 [syringic acid (SA)−H]⁻, 182.021 [(SA−H)−CH₃]•⁻ and 153.055 [(SA−H)−CO₂]⁻. Compounds 49 and 50 were identified as isomeric hydroxydihydrocaffeoyl–syringoyl–hexaric acids (Table 1, Figure 4). Acylhexaric acids are reported for the first time in S. scardica.

Figure 4. MS/MS spectrum of (A) hydroxydihydrocaffeoyl–hexaric acid (47) and (B) hydroxydihydrocaffeoyl–siryngoyl–hexaric acid (49).

2.1.6. Phenylethanoid Glycosides

A class of secondary metabolites distinctive for Sideritis species were phenylethanoid glycosides [12]. The typical fragmentation pattern revealed the loss of 162.05, 146.05, 179.03, 18.01 Da—corresponding to glucosyl and rhamnosyl moieties—deprotonated caffeic acid, and H₂O, respectively. Detailed discussion on the MS/MS fragmentation has been previously provided [12]. Based on a comparison with literature data, 10 phenylethanoid glycosides were dereplicated in the studied S. scardica extract (Table 1).

2.1.7. Iridoid Glycosides

The characteristic loss of hexose (−162.05 Da) and 7-(hydroxymethyl)-4,5-dihydrocyclopentapyran-4,5-diol (−182.06 Da, C₉H₁₀O₄) indicated the presence of iridoid glycosides [12]. Compound 61 with deprotonated molecules at m/z 523.166 was dereplicated as melittoside [12]. Fragment ions at m/z 163.039, 179.034, and 193.050 corresponding to the deprotonated coumaric, caffeic, and ferulic acids, led to the identification of 62, 63, and 64 as p-coumaroylmelittoside, caffeoylmelitoside, and feruloylmelitosside, respectively. Compounds 63 and 64 were found for the first time in S. scardica (Table 1). Previously, 63 has been isolated from S. lanata [26], while 64 has been found in S. trojana [27].

2.1.8. Flavonoids

Flavonoids are the dominant secondary metabolites in Sideritis species [12]. The main flavonoids in the studied species were O-glycosides of the flavones isoscutellarein ([Agl−H]⁻ at m/z 285.041), methylisoscutellarein ([Agl−H]⁻ at m/z 299.056), hypolaetin ([Agl−H]⁻ at m/z 301.036), methylhypolaetin ([Agl−H]⁻ at m/z 315.052), and apigenin ([Agl−H]⁻ at m/z 285.041). The MS/MS fragmentation patterns of the glycosides have been described previously [12] and are based on the loss of 162.054, 324.106, and 42.016 Da, corresponding to O-hexose/dihexose/acetyl group, respectively. The occurrence of a 180 Da loss [M−H-hex-H₂O]⁻ was indicative of 1→2 glycosylation between two sugars [16]. Significant fragments in the flavone aglycone annotation were a series of neutral losses of CO (−28 Da), CO₂ (−44 Da), CH₂O (−30 Da), and H₂O (−18 Da), supported by the retro-Diels–Alder (rDA) cleavages ^0,4A⁻, ^1,2A⁻, ^1,3A⁻, ^1,2B⁻, and ^1,3B⁻ [22,23]. In general, five glycosides of isoscutellarein (IS) (70, 71, 72, 78, 83), three of methylisoscutellarein (MIS) (80, 82, 88), two of hypolaetin (HL) (67, 73), four of methylhypolaetin (MHL) (74, 76, 79, and 84), five of apigenin (Api) (65, 69, 75, 77, 86), and one of luteolin (Lu) (66) were dereplicated in the S. scardica extract (Table 1). Apigenin and luteolin were evidenced by the rDA ions at m/z 151.002 (^1,3A⁻), 107.012 (^0,4A⁻), 117.033 (^1,3B⁻) (Api), and 133.028 (^1,3B⁻) (Lu). The aglycones isoscutellarein and hypolaetin were deduced from the rDA cleavages ^1,2A⁻-H₂O at m/z 163.003, ^1,3A⁻-CO at m/z 136.986, as well as ^1,3B⁻ at m/z 117.033 (IS) and ^1,3B- at m/z 133.028 (HL). Their methoxylated derivatives revealed a fragment ion [Agl−H−CH₃]•⁻ at m/z 284.033 (MIS) and 300.028 (MHL). Compound 70 ([M−H]⁻ at m/z 579.136) gave a base peak at m/z 285.041 [M−H−pent-hex]⁻. Thus, 70 was related to isoscutellarein O-pentosylhexoside. Illustrations of the fragmentation pathways of glycosides of the four abovementioned flavones aglycons are presented in Figure 5. The flavanone naringenin [M−H]⁻ at m/z 271.061 (85) was identified based on the RDA fragments at m/z 151.002 (^1,3A⁻), 107.012 (^0,4A⁻), and 119.049 (^1,3B⁻). In addition, its dihexoside (68) and coumaroylhexoside (87) were annotated based on the neutral loss of two hexoses and coumaroylhexose, respectively. Compounds 68, 70, 85, and 87 were annotated for the first time.

Figure 5. Fragmentation pathways for flavones aglycons caused by cleavage of C-ring bonds in negative ion mode.

2.1.9. Fatty Acids and Organosulfur Compounds

A saturated (98), two monounsaturated (94 and 97), and four polyunsaturated (93, 95, 96, and 99) fatty acids were tentatively identified in S. scardica extract. Among them, the main fatty acids were trihydroxyoctadecadienoic acid (93) and trihydroxyoctadecenoic acid (94), previously reported for the species [12]. The dereplication and fragmentation pathway of dihydroxy fatty acids have been previously described [28]. In addition, four organosulfur compounds (100–103) were dereplicated based on fragment ions at m/z 96.959 [HO₄S]⁻ and 79.956 [O₃S]⁻. These compounds have been previously described for S. scardica [12].

Table 1. UHPLC–HRMS metabolite profiling of Sideritis scardica infusion with content (mg/g lyophilized infusion) of compounds assayed.

No	Identified/Tentatively Annotated Compound	Molecular Formula	Exact Mass [M−H]⁻	Fragmentation Pattern in (-) ESI-MS/MS	tR (min)	Δ ppm	Level of Confidence [29]	Content [mg/g li] Mean ± SD
Sugar acids and saccharides
1.	Xylonic acid	C₅H₁₀O₆	165.0405	165.0395 (60.5), 147.0288 (8.8), 129.0181 (12.3), 111.0073 (0.3), 105.0179 (33.1), 101.0228 (2.7), 87.0073 (43.1), 75.0072 (100)	0.68	−6.006	2	-
2.	Hexose	C₆H₁₂O₆	179.0561	179.0541 (68.6), 161.0444 (11.1), 143.0338 (10.1), 125.0231 (1.53), 99.0437 (1.71), 81.0331 (4.8), 75.0072 (100)	0.70	−4.975	2	-
3.	Gluconic acid	C₆H₁₂O₇	195.0510	195.0503 (96.1), 177.0396 (18.6), 159.0288 (14.7), 147.0287 (15.5), 141.0184 (5.9), 129.0180 (48.3), 111.0073 (5.3), 105.0179 (332.8), 75.0072 (100)	0.72	−3.619	2	-
4.	Pentose	C₅H₁₀O₅	149.0456	149.0444 (21.6), 131.0334 (5.2), 101.0224 (0.8), 89.0229 (17.8), 75.0072 (100)	0.74	−7.358	2	-
5.	Asystoside	C₂₅H₄₄O₁₅	583.2607	583.2618 (100), 421.2090 (4.9), 289.1662 (15.4), 161.0445 (15.8), 101.0230 (21.3), 71.0123 (30.6)	6.34	1.880	2	-
Carboxylic acids
6.	Quinic acid	C₇H₁₂O₆	191.0561	191.0553 (100), 173.0446 (1.8), 155.0340 (0.2), 137.022 (0.2), 127.0387 (3.3), 99.0438 (0.6), 93.0331 (5.6), 85.0279 (18.2), 71.0123 (1.6), 59.0123 (1.37)	0.69	−4.194	2	-
7.	Oxaloglutaric acid	C₇H₈O₇	203.0197	203.0191 (100), 159.0292 (2.3), 141.0181 (27.8), 115.0022 (11.7), 97.0279 (97.1), 95.0123 (14.2), 79.0174 (11.2), 72.9915 (7.1), 71.0123 (50.6), 69.0330 (66.2)	0.88	−2.984	2	-
8.	Citric acid	C₆H₈O₇	191.0197	191.0191 (2.6), 173.0084 (1.43), 154.9979 (0.7), 147.0286 (0.4), 129.0181 (6.1), 111.0074 (100), 101.0231 (0.7), 87.0073 (43.8), 85.0280 (27.0)	0.90	−3.119	2	-
Hydroxybenzoic, hydroxycinnamic acids, and their derivatives
9.	Gallic acid	C₇H₆O₅	169.0143	169.0133 (37.5), 125.0231 (100), 97.0280 (3.8), 69.0330 (4.9)	1.13	−5.660	1	3.45 ± 0.51
10.	Hydroxybenzoic acid O-pentosylhexoside ^a	C₁₈H₂₄O₁₂	431.1194	431.1200 (69.3), 299.0776 (2.3), 137.0232 (100), 93.0331 (73.0)	1.78	1.231	2	1.32 ± 0.13
11.	Vanillic acid O-pentosylhexoside ^a	C₁₉H₂₆O₁₃	461.1301	461.1310 (76.2), 329.0879 (1.3), 167.0340 (100), 152.0104 (52.4), 123.0438 (11.3), 108.0203 (27.4)	1.99	2.052	2	2.27 ± 0.35
12.	Protocatechuic acid ^a	C₇H₆O₄	153.0193	153.0182 (15.9), 123.0439 (0.1), 109.0281 (100), 81.0330 (1.4), 65.0380 (0.38)	2.01	−7.397	1	7.98 ± 0.54
13.	Caffeic acid O-rutinoside (swertiamacroside)	C₂₁H₂₈O₁₃	487.1457	487.1465 (28.9), 179.0342 (100), 161.0234 (14.4), 135.0439 (45.5), 113.0232 (2.9)	2.80	1.572	2	4.68 ± 0.45
14.	Caffeic acid O-dihexoside ^a	C₂₁H₂₈O₁₄	503.1406	503.1414 (34.7), 341.0878 (42.9), 179.0341 (100), 135.0438 (77.1)	2.82	1.513	2	-
15.	2,3-Dihydroxybenzoic acid ^a	C₇H₆O₄	153.0193	153.0182 (51.9), 123.0074 (26.7), 108.0203 (100), 95.0124 (32.1), 85.0280 (33.1)	2.95	−7.267	2	-
16.	p-Hydroxybenzoic acid ^a	C₇H₆O₃	137.0244	137.0231 (35.2), 108.0200 (3.5), 93.0331 (100)	2.97	−9.614	1	-
17.	Dihydrocaffeic acid ^a	C₉H₁₀O₄	181.0506	181.0499 (52.1), 163.0377 (0.4), 137.0596 (100), 135.0439 (19.6), 123.0436 (58.0), 121.0282 (24.9), 119.0489 (15.1), 109.0281 (27.6), 93.0332 (2.5), 59.0124 (86.3)	3.33	−4.154	2	-
18.	2,4-Dihydroxybenzoic acid ^a	C₇H₆O₄	153.0193	153.0182 (76.8), 135.0075 (29.7), 123.0439 (0.26), 109.0281 (100), 108.0201 (0.2), 91.0174 (5.6), 81.0333 (0.3), 65.0381 (14.5)	3.47	−7397	1	4.77 ± 0.74
19.	p-Hydroxyphenyl acetic acid ^a	C₈H₈O₃	151.0400	151.0389 (100), 136.0156 (20.1), 123.0075 (4.6), 109.0283 (11.5), 107.0489 (2.3)	3.49	−7.133	1	-
20.	Caffeic acid	C₉H₈O₄	179.0350	179.0341 (21.3), 135.0439 (100), 117.0335 (0.7), 107.0487 (1.35), 91.0537 (0.5)	3.54	−4.759	1	87.25 ± 6.54
21.	Gentisic acid	C₇H₆O₄	153.0193	153.0182 (45.9), 109.0281 (100), 91.0175 (1.1), 108.0203 (8.7), 81.0330 (1.9), 65.0382 (0.1)	3.65	−7.397	1	0.42 ± 0.05
22.	Ferulic acid O-rutinoside ^a,b	C₂₂H₃₀O₁₃	501.1614	501.1618 (10.2), 193.0500 (100), 175.0393 (8.4), 160.0157 (9.7), 134.0361 (43.4), 113.0230 (5.5)	3.78	3.125	2	-
23.	p-Coumaric acid O-hexoside ^a	C₁₅H₁₈O₈	325.0928	163.0391 (100), 145.0288 (4.14), 119.0488 (35.7)	3.81	3.197	2	-
24.	Syringic acid ^a	C₉H₁₀O₅	197.0455	197.0449 (40.0), 182.0213 (48.9), 153.0547 (100), 138.0311 (11.5), 123.0075 (26.7), 121.0281 (85.6), 106.0046 (9.67), 95.0123 (8.1), 89.0018 (14.97)	4.34	−3.231	2	1.69 ± 0.26
25.	O-coumaric acid ^a	C₉H₈O₃	163.0401	163.0390 (9.7), 135.0075 (0.2), 119.0489 (100)	4.55	−6.363	1	-
26.	Ferulic acid	C₁₀H₁₀O₄	193.0506	193.0499 (100), 178.0260 (14.1), 165.0545 (13.5), 149.0600 (21.4), 134.0358 (12.3), 123.0438 (92.0), 79.0538 (4.1)	5.17	−3.637	1	-
Caffeic acid oligomers
27.	Lithospermic acid ^a	C₂₇H₂₂O₁₂	537.1038	537.1035 (7.1), 493.1142 (30.4), 339.0515 (100), 313.0726 (8.8), 295.0613 (23.6), 267.0671 (10.9), 179.0345 (12.4), 135.0440 (49.3)	4.97	−0.743	2	0.51 ± 0.05
28.	Salvianolic acid B ^a	C₃₆H₃₀O₁₆	717.1460	717.1478 (49.4), 673.1570 (7.7), 537.1055 (28.0), 519.0946 (47.9), 493.1153 (5.9), 339.0512 (13.0), 321.0409 (26.0), 313.0717 (11.5), 295.0616 (100), 277.0513 (6.7), 229.0141 (8.7), 203.0346 (13.9), 197.0447 (2.5), 179.0340 (11.3), 161.0237 (1.1), 135.0439 (32.2), 109.0281 (71.9)	6.06	2.401	2	1.42 ± 0.01
29.	Rosmarinic acid	C₁₈H₁₆O₈	359.0772	359.0782 (14.1), 197.0449 (26.0), 179.0342 (11.3), 161.0233 (100), 135.0439 (14.64), 133.0282 (19.8), 109.0275 (0.4)	6.33	2.56	1	6.07 ± 0.46
30.	Isosalvianolic acid C ^a	C₂₆H₂₀O₁₀	491.0983	491.0991 (100), 311.0567 (98.4), 267.0666 (39.3), 265.0508 (3.9), 249.0559 (1.5), 197.0454 (2.2), 179.0339 (1.8), 135.0440 (48.7)	7.29	1.466	2	0.42 ± 0.03
31.	Didehydrosalvianolic acid B ^a	C₃₆H₂₈O₁₆	715.1305	715.1324 (52.4), 535.0894 (20.2), 517.0786 (5.4), 337.0357 (8.1), 319.0241 (12.4), 311.0575 (7.6), 293.0461 (100), 265.0503 (7.1), 197.0446 (8.9), 135.0438 (10.5), 109.0279 (5.5)	7.76	2.786	2	0.19 ± 0.03
Acylquinic acids
32.	(Neo)chlorogenic acid O-hexoside ^a	C₂₂H₂₈O₁₄	515.1406	515.1414 (52.2), 353.0883 (5.6), 191.0553 (100), 179.0351 (3.4), 135.0441 (4.0), 93.0332 (11.0)	2.13	1.536	2	-
33.	Neochlorogenic acid	C₁₆H₁₈O₉	353.0877	353.0883 (40.7), 191.0555 (100), 179.0341 (61.2), 173.0451 (3.1), 161.0235 (3.5), 135.0439 (48.7), 93.0331 (4.5)	2.36	0.575	1	-
34.	Chlorogenic acid O-hexoside ^a	C₂₂H₂₈O₁₄	515.1406	515.1414 (100), 323.0767(51.9), 191.0554 (94.8), 179.0327 (4.5), 161.0238 (33.5), 135.0434 (6.4), 111.0435 (4.0)	2.85	2.487	1	-
35.	3-p-coumaroylquinic acid ^a	C₁₆H₁₈O₈	337.0928	337.0932 (6.9), 191.0555 (6.9), 173.0449 (3.5), 163.0390 (100), 135.0437 (0.5), 119.0488 (26.5), 111.0438 (0.7), 93.0332 (0.8)	2.99	0.918	2	-
36.	Chlorogenic acid	C₁₆H₁₈O₉	353.0877	353.0879 (4.6), 191.0554 (100), 179.0380 (0.9), 161.0235 (1.9), 111.0437 (0.8), 93.0331 (2.7), 85.0280 (7.5)	3.17	0.325	1	5.22 ± 0.21
37.	5-Syringoylquinic acid ^a	C₁₆H₂₀O₁₀	371.0983	371.0987 (36.1), 197.0448 (4.9), 191.0554 (100), 173.0443 (14.6), 153.0538 (2.3), 121.0279 (9.4), 111.0435 (2.8), 93.0331 (31.6), 85.0279 (3.7)	3.30	0.862	3	-
38.	4-Caffeoylquinic acid ^a	C₁₆H₁₈O₉	353.0877	353.0883 (32.7), 191.0555 (39.50), 179.0342 (75.3), 173.0447 (100), 135.0439 (52.1), 111.0436 (3.3), 93.0331 (21.3)	3.36	0.515	2	7.65 ± 0.96
39.	3-Feruloylquinic acid	C₁₇H₂₀O₉	367.1034	367.1036 (19.5), 193.0500 (100), 173.0447 (4.7), 137.0226 (3.4), 134.0361 (56.7)	3.42	0.339	2	0.63 ± 0.04
40.	4-Syringoylquinic acid ^a	C₁₆H₂₀O₁₀	371.0983	371.0978 (9.8), 197.0456 (11.5), 191.0554 (100), 173.0443 (14.6), 153.0538 (2.3), 121.0283 (4.9), 111.0435 (2.8), 93.0331 (14.5)	3.43	−1.509	3	-
41.	5-p-Coumaroylquinic acid ^a	C₁₆H₁₈O₈	337.0928	337.0940 (8.9), 191.0554 (100), 173.0446 (6.6), 163.0390 (5.31), 119.0488 (4.8), 111.0437 (2.0), 93.0331 (16.7)	3.96	3.172	2	1.85 ± 0.03
42.	4-p-Coumaroylquinic acid ^a	C₁₆H₁₈O₈	337.0928	337.0941 (8.6), 191.0555 (2.7), 173.0445 (100), 163.0390 (18.5), 119.0488 (9.0), 111.0436 (3.1), 93.0330 (22.2)	4.02	3.558	2	1.82 ± 0.05
43.	5-Feruloylquinic acid	C₁₇H₂₀O₉	367.1034	367.1038 (23.7), 193.0500 (16.9), 191.0554 (100), 173.0446 (81.4), 155.0340 (3.8), 134.0361 (19.2), 111.0438 (4.9), 93.0331 (39.7)	4.38	0.912	2	5.13 ± 0.08
44.	1-p-Coumaroylquinic acid ^a	C₁₆H₁₈O₈	337.0928	337.0932 (7.2), 191.0554 (100), 173.0445 (100), 135.0447 (2.3), 163.0393 (0.5), 119.0487 (1.1), 111.0437 (0.5), 93.0331 (5.3)	4.60	1.007	2	0.56 ± 0.04
45.	1-Feruloylquinic acid	C₁₇H₂₀O₉	367.1034	367.1039 (10.2), 191.0554 (100), 179.0340 (0.5), 173.0448 (2.3), 161.0239 (0.3), 134.0360 (3.1), 111.0440 (1.41), 93.0331 (5.15)	4.90	−2.996	2	1.12 ± 0.16
46.	Syringoyl–caffeoylquinic acid ^a	C₂₅H₂₆O₁₃	533.1300	533.1305 (14.5), 335.0777 (44.6), 291.0875 (20.7), 197.0450 (100), 153.0546 (12.6), 137.0232 (38.4), 123.0073 (17.0), 111.0439 (10.4), 93.0331 (49.0)	6.90	0.799	2	0.81 ± 0.08
Acylhexaric acids
47.	Hydroxydihydrocaffeoyl–hexaric acid ^a,b	C₁₅H₁₈O₁₂	389.0725	389.0730 (16.8), 371.0631 (0.8), 209.0297 (16.8), 191.0192 (34.2), 153.0539 (1.0), 147.0286 (17.9), 129.0180 (10.8), 85.0280 (100)	1.65	1.236	3	1.12 ± 0.16
48.	Hydroxydihydrocaffeoyl–hexaric acid isomer ^a,b	C₁₅H₁₈O₁₂	389.0725	389.0728 (16.8), 371.0621 (2.7), 209.0294 (17.2), 197.0442 (3.7), 191.0191 (45.3), 173.0087 (3.1), 147.0286 (20.6), 129.0180 (13.5), 111.0079 (4.5), 85.0280 (100)	2.60	0.773	3	0.81 ± 0.08
49.	Hydroxydihydrocaffeoyl–siryngoyl–hexaric acid ^a,b	C₂₄H₂₆O₁₆	569.1148	569.1159 (49.1), 389.0726 (8.8), 371.0627 (52.7), 327.0726 (13.8), 209.0299 (1.4), 197.0450 (39.1), 191.0186 (3.7), 182.0211 (3.3), 173.0084 (18.4), 166.9975 (1.6), 147.0285 (10.0), 138.0309 (1.7), 129.0181 (54.5), 123.0072 (1.6), 121.0282 (17.5), 111.0073 (14.3), 97.6908 (1.7), 85.0280 (100)	3.53	1.937	3	1.77 ± 0.15
50.	Hydroxydihydrocaffeoyl–siryngoyl–hexaric acid isomer ^a,b	C₂₄H₂₆O₁₆	569.1148	569.1158 (43.9), 389.0731 (7.6), 371.0625 (42.8), 327.0728 (12.2), 209.0295 (1.13), 197.0450 (40.8), 191.0186 (3.8), 182.0214 (4.3), 173.0084 (15.4), 166.9978 (1.0), 153.0548 (10.2), 147.0288 (8.0), 138.0315 (2.2), 129.0181 (59.1), 123.0074 (3.6), 121.0281 (16.5), 111.0073 (12.5), 85.0280 (100)	3.77	2.148	3	3.53 ± 0.237
Phenylethanoid glycosides
51.	Decaffeoyl aceteoside/verbasoside	C₂₀H₃₀O₁₂	461.1664	461.1673 (100), 315.1085 (5.2), 297.0984 (2.6), 135.0439 (29.4), 113.0230 (46.0), 85.0280 (20.6), 71. 0123 (20.7)	2.57	0.881	2	35.09 ± 2.46
52.	Hydroxyverbascoside	C₂₉H₃₆O₁₆	639.1931	639.1946 (86.8), 621.1832 (5.3), 459.1533 (1.9), 179.0342 (36.0), 161.0232 (5.3), 135.0440 (22.3), 133.0283 (37.6), 113.0231 (8.6)	4.49	2.381	2	13.45 ± 0.47
53.	Verbascoside	C₂₉H₃₆O₁₅	623.1987	623.1996 (60.3), 461.1674 (9.3), 315.1078 (1.80), 179.0340 (2.4), 161.0234 (100), 135.0440 (8.7), 133.0283 (23.9)	5.48	2.305	2	151.54 ± 10.86
54.	Echinacoside	C₃₅H₄₆O₂₀	785.2509	785.2529 (72.5), 623.2175 (7.7), 461.1666 (6.3), 179.0349 (3.6), 161.0234 (100), 135.0440 (24.9), 133.0282 (47.0)	5.23	2.424	2	0.74 ± 0.005
55.	Forsythoside B/samioside/lavandulifolioside	C₃₄H₄₄O₁₉	755.2403	755.2424 (80.5), 593.2080 (8.2), 461.1675 (8.7), 267.1614 (1.5), 179.0341 (6.8), 161.0234 (100), 135.0439 (23.5), 133.0282 (45.8), 113.0231 (9.4)	5.38	2.606	2	6.57 ± 0.103
56.	Alyssonoside	C₃₅H₄₆O₁₉	769.2560	769.2578 (100), 593.2076 (8.5), 461.1658 (8.9), 193.05 (20.9), 175.0393 (44.1), 161.0236 (10.2), 160.0156 (44.4), 135.0442 (15.4), 134.0362 (20.9), 132.0206 (14.9), 123.0439 (7.4), 113.023 (15.1), 85.0281 (9.3), 71.0124 (10.0)	6.07	22.207	2	2.16 ± 0.14
57.	Leucoseptoside A	C₃₀H₃₈O₁₅	637.2138	637.2154 (100), 461.1669 (16.2), 315.1091 (3.8), 193.0501 (13.00), 175.0392 (67.8), 161.0233 (109), 160.0155 (60.1), 113.0230 (18.9)	6.25	2.505	2	22.80 ± 0.82
58.	Leontoside B/stachyoside D	C₃₆H₄₈O₁₉	783.2716	783.2734 (100), 193.0501 (34.7), 175.0392 (92.4), 167.0700 (1.5), 160.0156 (73.8), 132.0205 (26.8)	6.97	2.142	2	0.90 ± 0.05
59.	Acetylverbascoside	C₃₁H₃₈O₁₆	665.2087	665.2103 (65.0), 503.1778 (3.3), 461.1676 (3.1), 161.0234 (100), 179.0339 (2.9), 135.0440 (10.4), 133.0283 (38.4), 113.0229 (1.9)	6.99	2.348	2	0.90 ± 0.01
60.	Martynoside	C₃₁H₄₀O₁₅	651.2294	651.2317 (95.8), 475.1836 (1.2), 329.1252 (1.1), 193.0500 (13.6), 175.0392 (100), 160.0156 (69.1), 132.0204 (25.1), 113.0230 (14.0)	7.21	3.450	2	11.35 ± 0.75
Iridoid glycosides
61.	Melittoside	C₂₁H₃₂O₁₅	523.1668	523.1676 (10.9), 361.1139 (4.5), 343.1040 (5.4), 325.0919 (1.3), 313.9523 (0.4), 283.0820 (0.5), 253.0722 (1.3), 223.0613 (1.7), 205.0506 (1.6), 179.0553 (100), 161.0447 (16.8), 119.0337 (34.9), 101.0230 (30.8), 89.0229 (80.9)	1.33	1.446	2	13.22 ± 1.36
62.	p-Coumaroylmelittoside	C₃₀H₃₈O₁₇	669.2035	669.2049 (100), 489.1423 (5.4), 325.0932 (28.3), 307.0823 (5.0), 265.0729 (3.2), 235.0605 (3.4), 205.0499 (16.9), 163.0390 (71.9), 145.0283 (84.4), 119.0488 (46.4), 93.0330 (4.9), 89.0230 (6.4)	3.98	1.909	2	3.71 ± 0.06
63.	Caffeoylmelittoside ^a,b	C₃₀H₃₈O₁₈	685.1974	685.1998 (100), 649.1036 (1.3), 523.1473 (1.9), 187.0396 (1.1), 181.0498 (24.9), 179.0342 (61.7), 163.0391 (39.9), 161.0235 (12.7), 135.0440 (78.4), 93.0331 (2.4), 89.0227 (2.3)	4.79	1.799	2	0.70 ± 0.01
64.	Feruloylmelittoside ^a,b	C₃₁H₄₀O₁₈	699.2142	699.2161 (75.9), 519.1519 (8.4), 357.0992 (54.0), 193.0500 (100), 163.0389 (30.6), 135.0435 (16.10), 134.0361 (70.0)	5.56	2.778	2	-
Flavonoids
65.	Apigenin 6,8-C-hexosyl hexoside	C₂₇H₃₀O₁₅	593.1522	593.1522 (100), 503.1212 (4.8), 473.1094 (14.5), 413.0891 (2.0), 395.0770 (0.8), 383.0776 (18.0), 353.0672 (30.1), 325.0729 (2.8), 297.0770 (10.7), 161.0235 (2.2), 117.0333 (3.3)	4.05	1.630	2	1.59 ± 0.18
66.	Luteolin 7-O-dihexoside	C₂₇H₃₀O₁₆	609.1461	609.1473 (100), 447.0949 (4.7), 285.0408 (61.9), 284.0331 (18.1), 256.0380 (1.0), 151.0025 (3.8), 133.0281 (3.4), 107.0121 (2.1)	4.94	2.023	2	0.58 ± 0.01
67.	Hypolaetin 7-O-hexosyl (1→2)-hexoside	C₂₇H₃₀O₁₇	625.1410	625.1422 (100), 463.0883 (6.6), 445.0775 (6.5), 301.0356 (88.8), 300.0279 (30.4), 283.0244 (0.7), 255.0292 (3.6), 227.0350 (2.3), 166.9975 (3.3), 163.0029 (1.1), 137.0232 (4.0), 133.0280 (7.5)	5.08	1.900	1	5.30 ± 0.09
68.	Naringenin 7-O-dihexoside	C₂₇H₃₂O₁₅	595.1668	595.1658 (100), 475.1172 (4.6), 355.0674 (5.6), 271.0619 (54.8), 270.0215 (1.8), 269.0457 (38.2), 151.0027 (43.4), 119.0488 (21.5), 107.0123 (18.3)	5.45	−1.703	2	-
69.	Apigenin 7-O-allosyl (1→2) glucoside	C₂₇H₃₀O₁₅	593.1512	593.1522 (64.7), 431.0986 (3.0), 269.0459 (100), 225.0546 (0.7), 151.0027 (1.4), 161.0232 (1.7), 117.0333 (3.3), 107.0125 (1.8)	5.50	1.731	2	1.93 ± 0.04
70.	Isoscutellarein 7-O-pentosyl–hexoside ^a,b	C₂₆H₂₈O₁₅	579.1356	579.1366 (38.9), 461.0086 (0.4), 285.0408 (100), 257.0451 (1.7), 241.0506 (1.6), 229.0497 (1.5), 213.0554 (5.1), 187.0393 (4.4), 136.9867 (0.9), 117.0330 (0.5)	5.51	1.825	2	1.01 ± 0.05
71.	Isoscutellarein 7-O-hexosyl (1→2)-hexoside	C₂₇H₃₀O₁₆	609.1461	609.1472 (87.2), 447.0912 (0.4), 429.0833(6.8), 285.0409 (100), 284.0328 (9.5), 255.0303 (2.2), 167.0496 (0.5), 163.0028 (2.1), 136.9868 (1.7), 117.0333 (1.8)	5.62	1.826	2	47.47 ± 0.95
72.	Isoscutellarein 7-O-hexoside	C₂₁H₂₀O₁₁	447.0933	447.0941 (26.4), 285.0408 (100), 229.0509 (1.0), 136.9868 (2.3), 117.0332 (1.1)	5.81	1.801	2	0.58 ± 0.01
73.	Hypolaetin 7-O-acetylhexosiyl–hexoside	C₂₉H₃₂O₁₈	667.1516	625.1406 (3.2), 463.0898 (3.2), 445.079 (6.6), 301.0357 (76.0), 300.0278 (30.8), 283.0285 (1.1), 255.0298 (1.4), 227.0347 (1.0), 166.9973 (1.9), 163.0020 (1.0), 137.0232 (4.0), 133.0284 (8.3), 109.0282 (1.0)	5.93	3.152	2	19.17 ± 1.07
74.	Methylhypolaetin 7-O-dihexoside	C₂₈H₃₂O₁₇	639.1567	639.1579 (75.3), 315.0516 (100), 300.0279 (40.7), 271.0264 (1.2), 243.0296 (2.5), 165.9901 (0.7), 136.9871 (6.2), 117.1944 (0.5), 133.0283 (2.5)	5.96	1.920	2	17.33 ± 0.25
75.	Apigenin 7-O-glucoside	C₂₁H₂₀O₁₀	431.0984	431.0988 (100), 269.0453 (24.9), 268.0381 (54.3), 211.0395 (1.6), 151.0025 (3.2), 117.0330 (1.8), 170.0124 (2.0)	6.06	1.044	1	0.46 ± 0.02
76.	Methylhypolaetin 7-O-hexoside	C₂₂H₂₂O₁₂	477.1039	477.1045 (30.5), 315.0515 (100), 300.0278 (32.0), 227.0350 (1.6), 136.9870 (6.2)	6.13	1.406	2	0.26 ± 0.01
77.	Apigenin 7-O-[6‴-O-acetyl]-hexosyl(1→2)-hexoside	C₂₉H₃₂O₁₆	635.1618	635.1629 (60.6), 593.1563 (1.1), 431.0981 (2.3), 269.0458 (100), 225.0560 (12.4), 151.0024 (1.7), 117.0332 (5.1), 107.0126 (2.6)	6.44	1.798	2	2.18 ± 0.003
78.	Isoscutellarein 7-O-hexosyl-(1→2)-[6″-O-acetyl]-hexoside	C₂₉H₃₂O₁₇	651.1567	651.1581 (69.3), 429.0831 (8.9), 285.0408 (100), 255.0285 (1.0), 239.0344 (1.1), 163.0026 (1.5), 136.9863 (0.8), 117.0334 (1.1)	6.58	2.161	2	151.70 ± 14.79
79.	4′-Methylhypolaetin 7-O-acetyl–hexosyl–hexoside	C₃₀H₃₄O₁₈	681.1672	681.1688 (89.6), 639.1533 (1.2), 357.0594 (0.9), 315.0516 (100), 300.0279 (44.2), 271.0248 (1.6), 243.0291 (1.4), 136.9868 (7.9), 133.0283 (4.1)	6.83	2.236	2	78.33 ± 3.29
80.	4′-Methylisoscutellarein 7-O-dihexoside	C₂₈H₃₂O₁₆	623.1618	623.1630 (100), 461.1117 (0.6), 299.0565 (83.6), 284.0330 (39.2), 255.0299 (3.0), 117.0330 (0.7)	7.23	1.929	2	24.20 ± 0.98
81.	Tremasperin	C₃₀H₃₄O₁₆	649.1774	649.1786 (5.4), 607.1672 (2.6), 283.0616 (100), 268.0381 (55.5), 284.0649 (5.8), 240.0431 (2.2), 151.0024 (0.5)	8.21	1.913	2	0.55 ± 0.04
82.	4′-O-methylisoscutellarein 7-O-[6‴-O-acetyl]hexosyl-(1→2)hexoside	C₃₀H₃₄O₁₇	665.1723	665.1740 (84.8), 299.0565 (100), 284.0330 (30.6), 255.0293 (2.5), 240.0429 (2.5), 227.0343 (2.5), 163.0025 (1.1), 136.9867 (9.2), 117.0338 (1.9)	8.24	2.447	2	107.44 ± 9.07
83.	Isoscutellarein 7-O-acetylhexosyl-O-acetylhexoside	C₃₁H₃₄O₁₈	693.1672	693.1663 (85.8), 471.0903 (7.4), 285.0407 (100), 213.0551 (6.0), 163.0022 (4.5), 136.9864 (1.9), 117.0331 (3.4)	8.26	−1.323	2	-
84.	Methylhypolaetin 7-O-acetylhexosyl-O-acetylhexoside	C₃₂H₃₆O₁₉	723.1778	723.1794 (89.7), 315.0515 (100), 300.0280 (44.9), 271.0255 (1.1), 243.0298 (1.4), 199.0390 (4.6), 136.9866 (9.5), 133.0284 (5.9)	8.47	2.182	2	0.31 ± 0.02
85.	Naringenin	C₁₅H₁₂O₅	271.0612	271.0615 (100), 227.0701 (0.7), 165.0180 (2.5), 151.0025 (65.7), 125.0228 (1.3), 119.0489 (52.3), 107.0124 (15.5), 93.0331 (11.9)	8.60	1.193	2	-
86.	Apigenin 7-O-p-coumaroyl-O-hexoside	C₃₀H₂₆O₁₂	577.1352	577.1360 (100), 431.0990 (13.4), 413.0890 (7.9), 269.0459 (77.0), 145.0283 (83.4), 163.0391 (4.4), 117.0332 (38.3), 107.0121 (1.1), 151.0026 (2.1)	9.06	1.457	2	0.75 ± 0.01
87.	Naringenin 7-O-coumaroylhexoside ^a	C₃₀H₂₈O₁₂	579.1508	579.1514 (100), 415.1033 (3.9), 307.0829 (12.0), 271.0616 (79.4), 151.0026 (40.9), 163.0391 (15.7), 145.0283 (57.4), 119.0489 (40.9), 107.0125 (15.2), 117.0332 (21.9)	9.15	0.985	2	1.09 ± 0.09
88.	4′-Methylisoscutellarein 7-O-(6‴-acetyl)-hexosyl(1→2)-[6′-O-acetyl]hexoside	C₃₂H₃₆O₁₈	707.1829	707.1844 (12.1), 299.0565 (100), 284.0330 (31.9), 300.0598 (6.8), 298.0496 (8.2), 255.0292 (4.7), 240.0424 (3.2), 227.0341 (1.7), 163.0023 (3.5), 136.9867 (10.7), 117.0332 (1.5)	9.89	2.125	2	0.09 ± 0.003
89.	Pectolinarigenin	C₁₇H₁₄O₆	313.0718	313.0721 (100), 298.0485 (53.9), 283.0251 (52.0), 269.0468 (2.4), 255.0302 (14.4), 227.0342 (2.17), 211.0386 (1.3), 183.0446 (2.1), 178.9918 (3.0), 163.0031 (11.8), 135.0075 (2.8), 117.0331 (13.1)	10.36	0.922	1	3.11 ± 0.50
90.	Eupatilin	C₁₈H₁₆O₇	343.0823	343.0826 (85.2), 328.0594 (100), 313.0360 (54.6), 298.0125 (13.3), 285.0412 (2.0), 270.0174 (42.6), 257.0095 (2.6), 133.0282 (3.7), 123.0439 (4.6)	11.05	0.915	2	0.47 ± 0.07
91.	8-Methoxycirsilineol	C₁₈H₁₆O₇	343.0823	343.0826 (100), 328.0594 (48.7), 313.0360 (71.8), 299.0952 (0.7), 298.0124 (209), 270.0175 (10.6), 242.0220 (4.2), 161.0233 (0.8), 117.0333 (8.9)	11.24	0.828	2	14.15 ± 2.30
92.	Genkwanin	C₁₆H₁₂O₅	283.0611	283.0615 (100), 268.0379 (67.4), 240.0428 (6.2), 239.0352 (1.8), 178.9915 (1.2), 151.0025 (4.1), 107.0125 (3.3)	11.42	1.036	2	-
Fatty acids
93.	Trihydroxyoctadecadienoic acid	C₁₈H₃₂O₅	327.2177	327.2166 (100), 309.2069 (0.8), 291.1971 (3.5), 229.1443 (12.5), 211.1334 (16.2), 183.1383 (1.6), 171.1015 (6.0), 85.0280 (2.5), 57.0329 (0.9)	9.15	0.986	2	-
94.	Trihydroxyoctadecenoic acid	C₁₈H₃₄O₅	329.2334	329.2338 (100), 311.2232 (1.4), 293.2119 (0.4), 229.1442 (17.2), 211.1335 (23.2), 183.1381 (2.7), 171.1020 (4.4), 127.1115 (1.6)	9.80	1.466	2	-
95.	Dihydroxyoctadecatrienoic acid	C₁₈H₃₀O₄	309.2071	309.2076 (100), 291.1972 (54.3), 247.2075 (1.0), 185.1179 (5.4), 137.0959 (17.9), 97.0645 (4.1)	10.90	1.641	2	-
96.	Dihydroxyoctadecadienoic acid	C₁₈H₃₂O₄	311.2228	311.2233 (100), 293.2132 (7.2), 275.2029 (6.4), 201.1128 (61.0), 183.1387 (1.6), 171.1015 (11.2), 127.1114 (4.3)12.62	12.62	1.662	2	-
97.	Dihydroxyoctadecenoic acid	C₁₈H₃₄O₄	313.2384	313.2389 (100), 295.2266 (5.8), 277.2166 (4.9), 201.1126 (42.8), 171.1012 (5.11), 127.1116 (4.8), 125.0960 (3.3)	13.75	1.460	2	-
98.	Dihydroxyoctadecanoic acid	C₁₈H₃₆O₄	315.2541	315.2544 (100), 297.2450 (4.3), 287.2241 (4.3), 171.1380 (0.6), 141.1272 (3.2), 127.1116 (0.6), 89.0230 (0.5)	14.87	1.101	2	-
99.	Hydroxylinoleic acid	C₁₈H₃₂O₃	295.2279	295.2282 (100), 277.2175 (14.6), 195.1384 (17.5), 113.0960 (1.1)	15.98	1.056	2	-
Organosulfur compounds
100.	Dodecyl sulfate	C₁₂H₂₆O₄S	265.1479	265.1482 (100), 96.9586 (66.4), 79.9558 (1.6)	14.55	1.081	2	-
101.	Lauryl ether sulfate	C₁₄H₃₀O₅S	309.1741	309.1746 (100), 122.9746 (1.9), 104.9527 (0.2), 96.9586 (54.3), 79.9558 (6.6)	16.10	1.624	2	-
102.	4-Dodecylbenenesulfonic acid	C₁₈H₃₀O₃S	325.1842	325.1846 (100), 216.0095 (0.2), 183.0113 (46.5), 197.0272 (0.8), 184.0147 (1.9)	17.44	0.957	2	-
103.	Myristyl sulfate	C₁₄H₃₀O₄S	293.1792	293.1796 (100), 96.9586 (73.6), 79.9558 (2.2)	17.89	1.455	2	-

^a—reported for the first time in the studied species; ^b—undescribed in the literature; level of confidence: 1—compound identified by comparison with reference standard; 2—putatively annotated compound; 3—putatively characterized compound classes.

2.2. Quantitative Determination

The quantitative determination of the main compounds in the profile of S. scardica lyophilized infusion was based on a common approach, where the HPLC analysis of the analytes was performed with a mobile phase composed of formic acid acetonitrile and water [22]. The content of the assayed compounds is revealed in Table 1. The main compounds in the tested lyophilized infusion were isoscutellarein-7-O-hexosyl-(1→2)-[6″-O-acetyl]-hexoside (78) and verbascoside (53), followed by 4′-O-methylisoscutellarein-7-O-[6‴-O-acetyl]hexosyl-(1→2)hexoside (82). Other dominant phenolic compounds include caffeic acid (20), 4′-methylhypolaetin-7-O-acetyl–hexosyl–hexoside (79), and isoscutellarein 7-O-hexosyl (1→2)-hexoside (71) (Table 1). Moreover, the data reveled moderate quantity of the phenylethanoid glycosides leucoseptoside A (57) and martynoside (60), as well as iridoid glycoside melittoside (61) (Table 1). The content of phenylethanoid glycosides ranged from 0.74 mg/g (54) to 151.54 mg/g lyophilized infusion (li) (53). With respect to caffeic acid oligomers, their quantities were found to range from 0.19 ± 0.033 mg/g li (31) to 6.07 ± 0.46 mg/g li (29), while caffeoylhexaric acids ranged from 0.81 ± 0.075 (48) to 3.53 ± 0.237 (50) (Table 1). However, this is the first attempt to quantify the secondary metabolites of the above classes in Sideritis species.

4-Caffeoylquinic acid (38) was found to be the dominant acylquinic acid (7.65 ± 0.96 mg/g li), followed by chlorogenic (36) and 5-feruloylquinic acid (43). Earlier quantitative research on the genus Sideritis showed that 5-caffeoylquinic acid has been found in all studied species as the most abundant hydroxycinnamic acid. In addition, the dominant phenolic compounds were the isoscutellarein derivatives isoscutellarein 7-O-[6‴-O-acetyl]-allosyl-(1→2)glucoside and 4′-O-methylisoscutellarein 7-O-allosyl-(1→2)-[6″-O-acetyl]-glucoside. Recently, eight compounds were detected in different Sideritis species: 5-caffeoylquinic acid, lavandulifolioside, verbascoside, isoscutellarein 7-O-allosyl(1→2)glucoside, hypolaetin 7-O-[6″-O-acetyl]-allosyl(1→2)glucoside, isoscutellarein 7-O-[6″-O-acetyl]-allosyl(1→2) glucoside, 3′-O-methylhypolaetin 7-O-[6″-O-acetyl]-allosyl(1→2)glucoside, 4′-O-methylhypolaetin, and 7-O-[6″-O-acetyl]-allosyl-(1→2)-[6″-O-acetyl]-glucoside). They represent 50% to 80% of the total phenolic content in S. scardica, S. raeseri, S. syriaca, and S. Taurica, and up to 90% in S. lanata [6]. The most abundant compounds present in the analyzed Siderits samples belonged to the group of phenylethanoid glycosides. The content of phenylethanoid glycosides ranged from 1.22 mg/g dry herb for S. lanata to 108.3 mg/g dry herb for S. scardica from Rhodopi Mountain, Bulgaria. The contribution of phenylethanoid glycosides to total phenolic content was around 50% for all samples, except for S. lanata where it accounted only for around 7% [6]. Eleven acetylated glycosides of isoscutellarein, hypolaetin, methylhypolaetin and methylisoscutellarein were previously isolated from 80% EtOH extract [30]. The differences between previous studies and our results can be ascribed to the different extraction methods and solvents.

2.3. Study Strength, Limitation and Future Direction

The study strength is that the presented extraction method of infusion is similar to the approach used in traditional medicine to process S. scardica tea. Therefore, this provides an insight into the phytochemical composition of common tea used in the traditional medicine and in-home remedies. A notable contribution of this study is the first-time dereplication and fragmentation patterns of five caffeic acids oligomers and four acylhexaric acids in S. scardica, expanding the current understanding of its chemical profile. The quantitative analysis identified major compounds in S. scardica infusion, with phenylethanoid verbascoside, glycosides of isoscutellarein, methylisoscutelarein, hypolaetin, and caffeic acid standing out as significant constituents. The reported concentrations add quantitative depth to the qualitative richness of the chemical composition. However, there are some limitations to the proposed method. In the quantitative assessment, a semi-quantitation was conducted, multiple detected substances were quantified based on a standard with a similar, yet different, chemical structure, as detailed above. Hence, a variation in the ionization between a standard and analytes may be a limitation. Future quantification based on the individual isolated secondary metabolites is recommended. In addition, isolation and accurate identification of the newly annotated caffeic acid oligomers and caffeoylhexaric acids will strengthen the validity of the present work.

3. Materials and Methods

3.1. Chemicals

Acetonitrile (hypergrade for LC–MS), formic acid (for LC–MS), and methanol (analytical grade) were purchased from Chromasolv (Sofia, Bulgaria). The reference standards used for compound identification were obtained from Extrasynthese (Genay, France) for protocatechuic, gentisic acids, and apigenin. Chlorogenic, caffeic, rosmarinic, cichoric acid, pectolinarigenin, and scutellarein were supplied from Phytolab (Vesten-bergsgreuth, Bavaria, Germany).

3.2. Plant Material

S. scardica seedlings were bought from a certified greenhouse “Mursalski-biogroup” (Bulgaria) and subsequently bred on alluvial soil with sunny exposure in an herbal garden (Rayanovtsi village, Vidin region) in Bulgaria at 349 m a.s.l. (43.7023° N 22.5206° E). The plant was identified by one of the authors (D.Z.) according to Assenov (1989) [31]. The plant material (aerial parts of 4-year-old plants) was collected during the flowering stage in July 2022 and dried for one week in the shade at room temperature. Then it was comminuted with a grinder (Rohnson, R-942, 220–240 V, 50/60 Hz, 200 W, Prague, Czech Republic) and stored in a dry and cool place until further analysis. The fresh/dried mass ratio is 4:1.

3.3. Sample Extraction

Air-dried aerial parts (100 g) were infused twice with boiled water (1:20 w/v) and extracted for 15 min at room temperature. The herbal infusion was lyophilized (lyophilizer Biobase BK-FD10P, BIOBASE, Jinan, China) to yield crude extracts of 12.5 g.

3.4. UHPLC–HRMS Dereplication/Annotation

The UHPLC–HRMS analyses were performed, as described previously [22], using a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) equipped with a heated electrospray ionization (HESI-II) probe (Thermo Scientific). The equipment was operated in negative ion mode within the m/z range from 130 to 2000 at a resolution of 70,000. Other instrument parameters for full MS mode were set as follows: automatic gain control (AGC) target 3 × 10⁶, maximum injection time (IT) 100 ms, number of scan ranges 1. For the DD-MS² mode, the instrument parameters were as follows: microscans 1, resolution 17,500, AGC target 1 × 10⁵, maximum IT 50 ms, MSX count 1, Top5, isolation window 2.0 m/z, stepped normalized collision energy (NCE) 10, 20, 60 eV. 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 containing 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The run time was 33 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. The equilibration time was 4 min. The injection volume and the flow rate were set to 1 µL and 300 µL/min, respectively. Data acquisition was performed using Xcalibur 4.2 (Thermo Scientific, Waltham, MA, USA) instrument control/data handling software.

3.5. UHPLC–HRMS Quantification

The UHPLC–HRMS quantification was conducted using the external standard method. Standard calibrations of protocatechuic (12), gentisic (21), caffeic (20), rosmarinic (27), chlorogenic (36), cichoric acids, apigenin, and scutellarein were established at five data points covering the concentration range of each analyte according to the level expected in the plant samples. Working solutions containing 0.05, 0.025, 0.012, 0.006, and 0.003 mg/mL of the assayed analytes were prepared from a stock solution in methanol containing 0.1 mg/mL. Based on the similar structure, the quantity of 9, 10, 14, and 21 was determined based on the calibration curve of gentisic acid; 11, 12, 18, and 24 as protocatechuic acid; 13, 14, 20, 51–56, and 63 as caffeic acid; 27–31 as rosmarinic acid; 36, 38, 39, 41–44 as chlorogenic acid; 65, 66, 69, 75, 77, 86–91 as apigenin; while 70–74, 76, 78–84, and 88 as scutellarein. Regression equations were as follows: gentisic acid y = 1,977,866,070x + 2460.9184 (R² = 0.9951) protocatechuic acid y = 1,416,556,589.51x + 1581.8936 (R² = 0.9965); caffeic acid y = 1,133,573,161x − 410.2916 (R² = 0.9928); rosmarinic acid y = 2,190,130,610x + 22,248.9144 (R² = 0.9950); chlorogenic acid y = 4,602,799,047.3258x − 921.0750 (R² = 0.9926); cichoric acid y = 4,589,524,443.3458x + 85.8499 (R² = 0.9999); apigenin y = 4,594,027,463.2779x + 21,088.0103 (R² = 0.9983); scutellarein y = 1,976,006,256x − 41,744.9522 (R² = 0.9420). The peak areas were calculated by integrating the Area Under the Curve (AUC) of the full-scan intensity scans for the corresponding molecular ion. These scans were also filtered for the presence of the characteristic base peak. MZmine 2.53 software was applied to the UHPLC–HRMS raw files of the studied S. scardica lyophilized infusion to obtain the peak area in the quantitative analysis. Results are expressed as mg/g lyophilized infusion.

4. Conclusions

In conclusion, an in-depth phytochemical analysis of S. scardica infusion using UHPLC–HRMS was performed. More than 100 metabolites, including sugar acids and saccharides, carboxylic, hydroxybenzoic, hydroxycinnamic, acylquinic and acylhexaric acids, caffeic acids oligomers, phenylpropanoid and iridoid glycosides, flavonoids, fatty acids, and organosulfur compounds were dereplicated/annotated. In addition, 62 metabolites of S. scardica were quantified. The presented extraction method of infusion is similar to the approach used in traditional medicine to process S. scardica tea. Therefore, the performed state-of-the-art phytochemical analysis of S. scardica provide additional knowledge with respect to the chemical constituents of this valuable medicinal plant.

Author Contributions

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

Funding

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.

Data Availability Statement

Data are contained within the article.

Acknowledgments

The authors are grateful to Stephan Mladenov, Faculty of Medicine, Medical University Sofia, for the supply of S. scardica plant material.

Conflicts of Interest

The authors declare no conflicts of interest.

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