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

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.


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].
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.

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)  , 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, Fig-

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 [ 1, Figure 4).Acylhexaric acids are reported for the first time in S. scardica.1, Figure 4).Acylhexaric acids are reported for the first time in S. scardica.

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 H 2 O, 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).

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.

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.

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.

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 × 10 6 , maximum injection time (IT) 100 ms, number of scan ranges 1.For the DD-MS 2 mode, the instrument parameters were as follows: microscans 1, resolution 17,500, AGC target 1 × 10 5 , 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
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.
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.

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

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