Metabolic Characterization of Four Members of the Genus Stachys L. (Lamiaceae)

Abstract

Several members of Stachys L. (among the largest Lamiaceae genera) have been traditionally used as medicinal plants. With 54 Stachys taxa (species and subspecies) occurring in mainland and/or insular Greece, the present study aimed to investigate the metabolic profiling of four range-restricted local Stachys members: Stachys candida and S. chrysantha (protected and endangered local Greek endemics), S. leucoglossa subsp. leucoglossa (local Balkan endemic), and S. spinulosa (local Balkan subendemic). In this investigation, the infusions of their above-ground parts were characterized using NMR and HPLC-PDA-MS techniques. Thus, 1D- and 2D-NMR spectra were obtained to compare the chemical fingerprints of these plants. Furthermore, previously isolated compounds from Stachys spp. were used to identify specific constituents. NMR screening revealed the presence of: (i) phenylethanoid glycosides, mainly acteoside in S. candida and S. chrysantha (section Candida, Swainsoniana phyloclade), and (ii) flavone 7-O-allosylglucoside (isoscutellarein 7-O-[6‴-O-acetyl-β-D-allopyranosyl]-(1→2)-β-D-glucopyranoside) and iridoids (monomelittoside or/and melittoside) in S. leucoglossa subsp. leucoglossa (section Olisia, Swainsoniana/Olisia phyloclade, Swainsoniana phyloclade) and caffeoylquinic acid (chlorogenic acid) in S. spinulosa (section Campanistrum, Stachys phyloclade). In total, 26 compounds were detected by HPLC-PDA-MS belonging to flavonoids, phenylethanoid glycosides, and phenolic acids. Among them, chlorogenic acid was identified in all samples as one of their main metabolites. The present study complements previous studies with first reports of constituents detected in the studied taxa, reports for the first time on the metabolic characterization of S. spinulosa, and discusses the chemotaxonomic significance of such findings.

1. Introduction

The genus Stachys L. (Lamiaceae family) comprises about 365 species, which are mainly distributed in the northern hemisphere [1,2,3] and are arranged in 19 sections [4] or different phyloclades [2]. In Greece, 54 species and subspecies of Stachys are distributed in parts of the mainland and/or insular country [5], and 41% of them are local endemics confined to Greece, while 28% are Balkan endemics or subendemics extending to Turkey and/or Italy.

Several Stachys taxa (species and subspecies) are used as infusions and decoctions in folk medicine, and their intended uses mainly concern the treatment of infections, the common cold, gastrointestinal disorders, inflammation, skin diseases, and wounds or as a remedy for asthma and anxiety implications [6]. Many of these folk uses (and related ancient or old recipes) date back to Dioscorides times in ancient Greece and Rome, traditional Chinese medicine, or Middle Eastern (Iranian and Turkish) folk phytotherapy [6]. Previous phytochemical investigations of Stachys spp. have reported several constituents belonging to different chemical categories, including terpenoids, iridoids, phytosterols, polyphenols (e.g., flavonoids, phenylethanoid glycosides, and phenolic acids), polysaccharides, and others [6,7]. Many Stachys taxa, as well as their isolated compounds, are known to exhibit diverse pharmacological properties, such as antioxidant, anti-inflammatory, anti-diabetic, anti-microbial, anti-proliferative, and cytotoxic activities, among others [6,7].

In our continuing endeavor to explore and document in phytochemical terms the different Stachys taxa growing in Greece [6], we have performed a chemical investigation herein in four members of the genus Stachys without bracteoles (or minute bracteoles), namely S. candida Bory & Chaub., S. chrysantha Boiss. & Heldr., S. leucoglossa Griseb. subsp. leucoglossa, and S. spinulosa Sm. More specifically, we focused on: (i) the endangered S. candida and S. chrysantha [8], which are local Greek (Peloponnese) endemics protected by the Greek Presidential Decree 67/1981 that belong to the section Candida [3,5] or the phyloclade Swainsoniana [2], with the first being an unarmed perennial with orbicular to ovate–orbicular leaves, calyx teeth longer than wide, and white corolla with purple spots, with the other being a non–spiny perennial with elliptic–ovate to suborbicular leaves and yellow tomentose corolla; (ii) the range-restricted local Balkan endemic S. leucoglossa subsp. leucoglossa belonging to the section Olisia or the Swainsoniana/Olisia phyloclade [2], an unarmed perennial with white or pale pink corolla in remote 2–6-flowered verticillasters with an almost glabrous calyx and very small floral leaves; and (iii) the local Balkan subendemic S. spinulosa which belongs to the section Campanistrum [3,5] or the Stachys phyloclade [2], a hispid annual with ± scabrid stem on angles, spiny bracts and crowded verticillasters in dense spikes, and coarsely spined cauline leaves, and lower leaves cordate at the base.

S. candida and S. chrysantha have been previously studied regarding their phytochemical composition, as well as the anti-inflammatory activity of their methanol extracts and their flavonoids [6]. Specifically, xanthomicrol, chrysoeriol, calycopterin, chrysoeriol-7-O-β-D-(3″-E-p-coumaroyl)-glucopyranoside, chrysoeriol-7-O-β-D-glucopyranoside, and isoscutellarein-7-O-[6‴-O-acetyl-β-D-allopyranosyl]-(1→2)-6″-O-acetyl-β-D-glucopyranoside were isolated from both plants, while luteolin-7-O-β-D-glucopyranoside was found only in S. chrysantha methanol extract. Additionally reported in S. candida were: four flavonoids (apigenin-7-O-β-D-glucopyranoside, isoscutellarein-7-O-[6‴-O-acetyl-β-D-allopyranosyl-(1→2)]-β-D-glucopyranoside, 4′-methyl-isoscutellarein-7-O-[6‴-O-acetyl-β-D-allopyranosyl-(1→2)]-β-D-glucopyranoside, and 4′-methyl-hypolaetin-7-O-[6‴-O-acetyl-β-D-allopyranosyl-(1→2)]-β-D-glucopyranoside), one phenylethanoid glycoside (acteoside), and one phenolic acid (chlorogenic acid). However, S. leucoglossa (not determined by subspecies) has been chemically characterized only once regarding its content in flavonoids (namely, isoscutellarein-7-O-[6‴-O-acetyl-β-D-allopyranosyl-(1→2)]-β-D-glucopyranoside, 4′-methyl-isoscutellarein-7-O-[6‴-O-acetyl-β-D-allopyranosyl-(1→2)]-β-D-glucopyranoside, and 4′-O-methyl-hypolaetin-7-O-[6‴-O-acetyl-β-D-allopyranosyl-(1→2)]-β-D-glucopyranoside) and iridoids (i.e., melittoside, harpagide, acetyl-harpagide, and ajugol) [9]. Nonetheless, S. spinulosa has not been subjected to any phytochemical study to date. Therefore, the present study aimed to investigate the metabolic profiles of the infusions of these four Greek Stachys taxa through NMR and HPLC-PDA-MS analyses.

2. Materials and Methods

2.1. Plant Material

The flowering above-ground parts of the studied plant species and subspecies were collected from wild-growing populations in different locations in Greece (Figure S1), and the living material was maintained ex situ at the premises of the Institute of Plant Breeding and Genetic Resources, Agricultural Organization Demeter (

Table 1. Investigated Stachys taxa (species and subspecies) with abbreviations used, sample origin, date of collections, and living or voucher specimen.

Species	Abbreviation	Sample Origin	Date	Living and/or Voucher Specimen
S. candida	SCA	Mt. Taygetos	29/5/2020	GR-1-BBGK-20,164-A
S. chrysantha	SCH	Mt. Taygetos	28/2/2020	GR-1-BBGK-20,96
S. leucoglossa subsp. leucoglossa	SLE	Mt. Karpouzi	13/6/2020	GR-1-BBGK-20,97
S. spinulosa	SSP	Sparta	29/5/2020	GR-1-BBGK-20,164-B

). Voucher specimens were identified by Dr. Nikos Krigas and deposited in the Herbarium of the Balkan Botanic Garden of Kroussia (BBGK), Institute of Plant Breeding and Genetic Resources, Agricultural Organization Demeter.

2.2. Preparation of the Infusions

Precisely 4.0 g of dried comminuted aerial parts of each plant sample was added to 200 mL of boiled water for 5 min, separately. Due to the fact that Stachys infusions in Greece have been traditionally prepared and consumed as ‘mountain tea’, referring to different Sideritis spp., the preparation of the infusions was based on the monograph of the European Medicines Agency (EMA) concerning different species and subspecies of the genus Sideritis [10]. Then, the samples were filtered and concentrated to dryness using a rota evaporator under reduced pressure to yield residues of 2.3 g for S. candida, 2.8 g for S. chrysantha, 2.0 g for S. leucoglossa subsp. leucoglossa, and 1.8 g for S. spinulosa. Distilled water was used as a solvent for the infusions, avoiding any additive/impurity in the samples.

2.3. NMR Analysis

In the NMR experiments, a part of each sample (5.0 mg) was dissolved in 600 μL of CD₃OD. The 1D- and 2D-NMR spectra of the samples were recorded on a Bruker 400 DRX instrument at 300 K. Chemical shifts are given in ppm (δ) and are referenced to the solvent signal at 3.31/ 49.0 ppm (CD₃OD) for ¹H- and ¹³C-NMR, respectively. COSY (COrrelation SpectroscopΥ) and HSQC (Heteronuclear Single Quantum Correlation) experiments were performed using standard Bruker microprograms.

2.4. HPLC-PDA-MS Analysis

HPLC-PDA-MS analysis was performed on a Thermo Finnigan system (Palo Alto, CA, USA), which consisted of an LC Pump Plus, Autosampler, and Surveyor PDA Plus Detector. The HPLC system was interfaced with an ESI MSQ Plus (Thermo Finnigan, San Jose, CA, USA) operating with Xcalibur software (version 2.1). The mass spectrometer operated in negative and positive ionization modes; the scan spectra were from m/z 100 to 1000, the gas temperature was at 350 °C, the nitrogen flow rate was at 10 L/min, and the capillary voltage was 3000 V. The cone voltage was in the range of 60–110 V. The column was an SB-Aq Zorbax (Agilent, Santa Clara, CA, USA) RP-C18 column (150 mm × 3.5 mm) with a particle size of 5 µm maintained at 30 °C. The eluents were H₂O at pH 2.8 by formic acid (0.05% v/v) (A) and acetonitrile (B) and with a flow rate of 0.4 mL/min. The gradient program was as follows: 0–7 min, 90–85%A; 7–12 min, 85–82%A; 12–25 82%A; 25–27 min, 82–75%A; 27–32 min, 75%A; 32–42 min, 75–60%A; 42–49 min, 60%A; 49–53 min, 60–90%A; 53–60 min, 90%. The injected volume of the samples was 5 μL of solution. The UV-vis spectra were recorded between 220 and 600 nm, and the chromatographic profiles were registered at 280, 330, and 350 nm.

3. Results and Discussion

The present study reports on the chemical fingerprints of the infusions of four Stachys taxa (S. candida, S. chrysantha, S. leucoglossa subsp. leucoglossa, and S. spinulosa) growing wild in Greece by means of NMR and HPLC-PDA-MS techniques. In general, LC–MS (Liquid Chromatography-Mass Spectrometry) and NMR (Nuclear Magnetic Resonance) are commonly used techniques for metabolic characterization in plants. By employing both techniques, the qualitative and quantitative strategy can be considerably improved, rendering the identification of plant extracts’ constituents feasible [11].

3.1. NMR Analysis

A preliminary screening of the Stachys spp. infusions was first obtained by ¹H-NMR spectra, and the chemical categories of their constituents were identified based on peaks in specific regions. The comparative 1D-NMR fingerprints are presented in Figure 1. In addition, 2D-NMR spectra (COSY and HSQC) were acquired to provide a better overview (Figures S1–S5). In all four ¹H-NMR spectra, signals of polysaccharides (region: 5.40–3.10 ppm) were noticed. However, some differences in the chemical fingerprints were observed among the samples.

In the ¹H-NMR spectra of S. candida (SCA) and S. chrysantha (SCH) belonging to the section Candida or the Swainsoniana phyloclade, mainly signals from phenylethanoid glycosides were detected (Figure 2A,B). Specifically, at δ_H 7.60 (d, J = 16.0 Hz) and 6.28 (d, J = 16.0 Hz), we found trans-coupling olefinic signals ascribable to double bonds (HSQC: δc 146.8 and 113.5, respectively), at the δ_H range of 7.07–6.57, we observed signals which could belong to protons of aromatic tri-substituted rings, and at δ_H around 2.80, we spotted signals of benzylic methylene protons (HSQC: δc 35.0). In the ¹H-¹H-COSY spectra of SCA and SCH, the principal correlation peaks among protons corresponding to phenylethanoid glycosides were also detected (Figures S2a and S3a). Their HSQC spectra are presented in Figures S2b and S3b. As an effort to interpret the observed different signals in the 1D-/2D-NMR spectra of SCA and SCH infusions, we compared them with the NMR spectra of previously isolated compounds sourced from our own previous works in Stachys spp. Through the careful screening of the ¹H-NMR spectra of different compounds, we noticed that the main proton signals in the spectra of S. candida and S. chrysantha infusions could be attributed to the phenylethanoid glycoside, namely acteoside. The overlaid ¹H-NMR spectra of both infusions and acteoside are illustrated in Figure 2. Acteoside has been previously found in several Stachys taxa [6,7], including S. candida [6]. Furthermore, the presence of flavones has been previously reported from members of the genus Stachys [6,7]. In Stachys species of the section Candida, chrysoeriol and isoscutellarein derivatives have been found [6]. It should be mentioned that minor signals of isoscutellarein derivatives were also detected in the SCH sample. However, it was not feasible to identify these constituents with previously isolated compounds in the NMR spectra due to signal overlapping and their low concentration.

In the ¹H-NMR spectrum of S. leucoglossa subsp. leucoglossa (SLE) belonging to the section Olisia or the Swainsoniana/Olisia phyloclade, mainly signals from flavonoids and iridoids were detected (Figure 3 and Figure 4). In the downfield region (δ_H 7.95–6.34), signals of flavones [δ_H: 7.94 (d, J = 8.7 Hz), 6.96 (d, J = 8.7 Hz), 6.79 (s), 6.66 (s)] were observed (Figure 3). In addition, a double peak (J = 6.5 Hz) appeared at δ_H 6.35. This assignment, along with the specific signals at δ_H 5.80 (s), 5.63 (d), and 5.10 (d), could belong to iridoids (Figure 4). In the ¹H-¹H-COSY spectrum, the principal correlation peaks among vicinal protons corresponding to flavones and iridoids were also detected (Figure S4a). The HSQC spectrum of SLE is presented in Figure S4b. By carefully screening the ¹H-NMR spectra of the SLE infusion with those of previously isolated compounds of Stachys spp., we noticed that the main proton signals in the spectrum of this infusion could be assigned to isoscutellarein derivatives, namely isoscutellarein-7-O-[6‴-O-acetyl-β-D-allopyranosyl]-(1→2)-β-D-glucopyranoside, as well as to monomelittoside or/and its 5-glucoside derivative, namely melittoside (Figure 3 and Figure 4). The presence of flavone 7-O-allosylglucosides has been previously reported from members of the genus Stachys [6,7]. In the Stachys species of the section Olisia, monoacetyl and diacetyl derivatives of isoscutellarein have been found, including reports of S. leucoglossa [6,9]. Furthermore, iridoids are known to be among the main metabolites in Stachys spp. [6,7], and previous studies have reported their presence in the members of the section Olisia, such as S. recta L. and S. spinosa L. [12,13], while melittoside has been found in S. leucoglossa [9].

In the ¹H-NMR spectrum of S. spinulosa (SSP) belonging to the section Campanistrum or the Stachys phyloclade, mainly signals from caffeoylquinic acid derivatives were detected (Figure 5). Specifically, at δ_H 7.58 (d, J = 16.2 Hz) and 6.28 (d, J = 16.2 Hz), we found trans-coupling olefinic signals ascribable to double bonds (HSQC: δc 147.0 and 115.7, respectively), and at the δ_H range of 7.05–6.77, the observed signals could belong to protons of aromatic tri-substituted rings. Although the signal overlapped in the middle area of the ¹H-NMR spectrum, signals of the oxymethine protons of the quinic acid moiety were spotted at δ_H 5.36 (HSQC: 72.3), 4.10 (HSQC: 73.0), and 3.67 (HSQC: 74.5), with its methylene protons also appearing at the δ_H range of around 2.15–1.95 (HSQC: δc 40.5). In the ¹H-¹H-COSY spectrum, the principal correlation peaks among protons of the caffeoyl and quinic acid moieties were detected (Figure S5a). The HSQC spectrum of SSP is presented in Figure S5b. By carefully screening the ¹H-NMR spectra of the SLE infusion with those of previously isolated compounds of Stachys spp., we noticed that the main proton signals in the spectrum of this infusion could be assigned to chlorogenic acid. The overlaid ¹H-NMR spectra of the SSP infusion and this compound are illustrated in Figure 5. Caffeoylquinic acids, especially chlorogenic acid, have been found in many Stachys species [6]. Moreover, flavones have been previously reported from two Stachys species belonging to the section Campanistrum [14,15]. Even though minor signals of flavones and other derivatives were also detected in the SSP sample, we were not able to identify specific constituents with previously isolated compounds in the NMR spectra due to signal overlapping and their low concentration. It should be mentioned that this is the first study addressing the phytochemical characterization of S. spinulosa.

3.2. HPLC-PDA-MS Analysis

In total, 26 compounds were detected by HPLC-PDA-MS (

Table 2. UV-VIS absorption and MS fragmentation data (positive and negative mode) of the compounds detected in the Greek Stachys infusions examined (SCA: Stachys candida; SCH: Stachys chrysantha; SLE: Stachys leucoglossa subsp. leucoglossa; SSP: Stachys spinulosa). The first reports of specific compounds for SCA, SCH, and SLE are indicated with an asterisk (*).

No.	Rt (min)	UV (nm)	Negative Mode, m/z	Positive Mode, m/z	Identification	SCA	SCH	SLE	SSP
1	10.90	298, 327	136.9, 172.9, 179.1, 191.1 [quinic acid-H]−, 353.1 [M-H]−, 707.1 [2M-H]−	354.9 [M+H]+	Chlorogenic acid isomer	+ *	+ *	+ *	+
2	11.20	298, 324	191.1 [quinic acid-H]−, 353.1 [M-H]−, 707.1 [2M-H]−	354.9 [M+H]+, 376.9 [M+Na]+	Chlorogenic acid	+	+ *	+ *	+
3	15.36	271, 334	353.1, 473.0, 592.9 [M-H]−	595.0 [M+H]+	Vicenin-2	+ *	-	-	-
4	22.28	291, 328	160.9, 593.2 [M-caffeoyl-H]−, 755.1 [M-H]−	479.1, 757.1 [M+H]+, 779.1 [M+Na]+	Lavandulifolioside (syn. Stachysoside A)	+ *	+ *	tr *	tr
5	23.73	291, 330	160.9 [caffeoyl group-H]−, 461.1 [M-caffeoyl-H]−, 623.1 [M-H]−	625.1 [M+H]+, 647.1 [M+Na]+	Acteoside	+	+ *	tr *	+
6	24.56	288, 329	160.9, 593.1, 623.2 [M-pentosyl-H]−, 755.2 [M-H]−	439.2, 625.1 [M-pentosyl+H]+, 757.0 [M+H]+, 779.1 [M+Na]+	Isomer of lavandulifolioside	+ *	+ *	-	-
7	27.26	289, 327	461.1 [M-caffeoyl-H]−, 623.0 [M-H]−	647.1 [M+Na]+	Acteoside isomer	+ *	+ *	-	-
8	28.57	289, 327	623.1 [M-H]−	647.1 [M+Na]+	Acteoside isomer	+ *	-	-	-
9	29.65	277, 307, 324	285.0 [A-H]−, 608.9 [M-H]−	611.0 [M+H]+	Isoscutellarein-7-O-allopyranosyl-(1→2)-glucopyranoside	-	-	+ *	-
10	32.03	288,329	137.0 [dihydroxytyrosol-H]−, 593.1 [M-feruloyl-H]−, 607.1 [M-rhamnose-H]−, 769.4 [M-H]−	771.1 [M+H]+, 793.2 [M+Na]+	Phenylethyl glycoside, isomer I (stachysoside B, syn. Leonoside A) tentatively	+ *	-	-	-
11	32.40	255, 268, 348	285.0 [A-H]−, 651.1 [M-H]−	653.1 [M+H]+, 661.1 [M+Na]+	Luteolin-acetyl-dihexoside	-	-	-	+
12	32.65–32.81	290, 329	136.9 [dihydroxytyrosol-H]−, 446.9, 637.1 [M-H]−	639.2 [M+H]+, 661.1 [M+Na]+	Leucosceptoside A	+ *	+ *	-	+
13	32.70	277, 307, 327	651 [M-H]−	653.1 [M+H]+	Isoscutellarein-7-O-[6‴-acetyl-allopyranosyl-(1→2)]-glucopyranoside Isomer I	-	-	+	-
14	33.06–33.20	253, 287, 296, 334	301.2 [A-H]−, 667.2 [M-H]−	479.1, 669.1 [M+H]+	Hypolaetin-acetylated derivative	+	+ *	+ *	+
15	33.44	268, 330	430.9 [M-H]−	433.1 [M+H]+	Apigenin-glucoside	+	-	-	-
16	33.66	282, 328	137.0 [dihydroxytyrosol-H]−, 476.9, 637.1, 769.1 [M-H]−	771.1 [M+H]+	Phenylethyl glycoside, isomer II (stachysoside B, syn. Leonoside A)	+*	+ *	-	-
17	34.05	267, 340 and 272, 287, 332			Mixture	+	-	-	-
18	34.62	277, 307, 327	651 [M-H]−	653.1 [M+H]+	Isoscutellarein-7-O-[6‴-acetyl-allopyranosyl-(1→2)]-glucopyranoside Isomer II	-	-	+	-
19	35.88	287, 329	783 [M-H]−	785.1 [M+H]+	Phenylethyl glycoside (stachysoside C) tentatively	+ *	-	-	-
20	35.99–36.21	276, 306, 328	285.0, [A-H]−, 429.1, 651 [M-H]−	653.1 [M+H]+	Isoscutellarein-7-O-[6‴-acetyl-allopyranosyl-(1→2)]-glucopyranoside	+	+	+	+
21	36.49	269, 342	298.9 [A-H]−, 623.1 [M-acetyl-H]−, 665.0 [M-H]−	667.1 [M+H]+	Chrysoeriol-7-O-acetyl-dihexoside (= stachyspinoside) tentatively	-	-	-	+
22	37.34–37.45	278, 296, 335	314.9 [A-H]−, 625.2 [M-acetyl-H]−, 681.1 [M-H]−	683.1 [M+H]+	3′-hydroxy-4′-O-methylisoscutellarein-7-O-[6‴-acetyl- allopyranosyl-(1→2)]-glucopyranoside	+	+ *	+	+
23	41.55–41.70	278, 297 338	300.9 [A-H]−, 709.0 [M-H]−	711.1 [M+H]+	hypolaetin-7-O-di-acetyl-dihexoside	-	+ *	+ *	+
24	42.24	277, 307, 322	298.9 [A-H]−, 665.0 [M-H]−	667.1 [M+H]+	4′-O-methylisoscutellarein-7-O-[6‴-acetyl-allopyranosyl-(1→2)]-glucopyranoside	-	-	+	tr
25	43.66	276, 307, 324	693.1 [M-H]−	695.1 [M+H]+	Isoscutellarein-7-O-diacetyl-dihexoside	-	+ *	+ *	tr
26	44.16–44.26	276, 297, 338	314.9 [A-H]−, 501.0 [M-hexosyl-acetyl-H2O-H]−, 723.1 [M-H]−	725.1 [M+H]+	3′-hydroxy-4′-O-methylisoscutellarein-7-O-diacetyl-dihexoside	-	+ *	+ *	+

A: aglycon; tr: traces.

), with them belonging to three main classes, namely flavonoids, phenylethanoid glycosides, and phenolic acids. The HPLC-PDA chromatograms of the Stachys infusions are illustrated in Figure S6.

Chlorogenic acid (2) was identified in all samples as one of the main metabolites in the Stachys infusions under study. The identification of the latter was based on a reference standard. An isobaric compound at an earlier retention time was assigned as an isomer, probably with a 4-substitution of the quinic acid group, as suggested by a fragment at m/z 173 [16]. Chlorogenic acid and isomers have previously been identified in the Stachys taxa of several sections, such as the sections Candida (S. candida and S. horvaticii Micevski, previously known as S. iva Griseb.), Eriostomum, and Olisia (S. atherocalyx K. Koch and S. recta L.), and members of the section Stachys [6]. However, this is the first report on the presence of chlorogenic acid and isomers in S. chrysantha, S. leucoglossa subsp. leucoglossa, and S. spinulosa.

Phenylethanoid glycosides are the main constituents of the genus Stachys [6,7]. In this study, nine phenylethanoid glycosides were identified in the four investigated infusions. Three detected phenylethanoid glycosides, namely lavandulifolioside (4), acteoside (5), and leucosceptoside A (12), were unambiguously identified by co-chromatography using compounds isolated in previous works by our group [6]. Although these compounds have previously been found in various Stachys taxa [6], they were detected for the first time in S. chrysantha, S. leucoglossa subsp. leucoglossa, and S. spinulosa. Acteoside has been previously isolated from S. candida [6], while lavandulifolioside and leucosceptoside A have not been identified in this species before. Peaks 6–8 were assigned tentatively as isomers of lavandulifolioside and acteoside as they had similar fragmentation patterns. Different substitution patterns of the acyl groups on the sugars might account for the differences in the retention times. Peaks 10 and 16 were tentatively assigned to stachysoside B and/or isomers, as suggested by the difference in the molecular weight (by 14 amu) when compared to lavandulifolioside. Furthermore, they were eluted at considerably longer retention times compared to lavandulifolioside. These compounds have been reported in S. affinis Bunge (synonym: S. sieboldii Miq.) [17]. Likewise, peak 19 was assigned tentatively to stachysoside C due to its pseudomolecular ion at m/z 783.0, suggesting the presence of two extra methyl groups in comparison to lavandulifolioside. The small amount of the latter in the infusion did not permit supporting further fragmentations. However, its retention time of almost 36 min agrees with this hypothesis. Such compounds are common in other Stachys species, such as S. affinis (synonym: S. sieboldii) [17] and S. plumosa Griseb. from Serbia [18].

The flavone glycosides detected in the infusions belong to the group of isoscutellarein/hypolaetin derivatives with some differentiations. Peaks 20, 22, and 24–26, being the main metabolites, were identified based on co-chromatography with previously isolated compounds from both laboratories [6]. Accordingly, peaks 9, 13, 14, 18, and 23 were identified either as their deacetylated counterparts or isomers resulting from different acylation sites or, ultimately, as diacetylated derivatives. Regarding the section Campanistrum, 8-hydroxyflavone-allosylglucosides have been found in S. arvensis (L.) L. and S. ocymastrum (L.) Briq. (=S. hirta L.) [15]. However, there is no previous report on such compounds in S. spinulosa. Regarding the section Candida, isoscutellarein/hypolaetin derivatives have been previously reported in S. candida, S. chrysantha, and S. horvaticii Micevski (previously known as S. iva) [6]. Furthermore, monoacetyl and diacetyl derivatives of isoscutellarein have been found in Stachys species of the section Olisia, including S. leucoglossa [6,9]. S. candida and S. spinulosa also contained apigenin/luteolin derivatives in small amounts. In a previous study, chrysoeriol and its derivative, namely chrysoeriol 7-(3″-E-p-coumaroyl)-D-glucopyranoside, were isolated from S. candida [6]. Vicenin-2 was identified by the diagnostic successive losses of 120 amu [19], while peak 15 was identified as apigenin-7-O-glucoside previously isolated from S. candida [6]. It should be mentioned that vicenin-2 and apigenin-7-O-glucoside were detected only in S. candida. In addition, this study reports the presence of vicenin-2 in this species for the first time. Peaks 11 and 21 were detected only in S. spinulosa and were identified as acetylated luteolin and chrysoeriol dihexosides based on UV and MS fragmentations as well as on previously isolated compounds from S. spinosa and S. aegyptiaca Pers. [13,19].

4. Conclusions

In the present study, the metabolic characterization of two protected and endangered local Greek endemic members of the genus Stachys (S. candida and S. chrysantha) and two local Balkan (sub-) endemic Stachys members (S. leucoglossa subsp. leucoglossa, and S. spinulosa) were investigated using NMR and HPLC-PDA-MS techniques. For the detection of specific constituents in the studied Stachys members, 1D- and 2D-NMR experiments were employed to compare their chemical fingerprints in combination with reference compounds isolated and identified in previous studies. In total, 26 compounds were detected by HPLC-PDA-MS as belonging to flavonoids (mainly isoscutellarein and hypolaetin derivatives), phenylethanoid glycosides, and phenolic acids (chlorogenic acid and its isomer). The chemical composition of S. spinulosa was investigated herein for the first time in detail, while knowledge of the metabolic profiling concerning the rest of the studied taxa was substantially supplemented herein with new reports of specific compounds not previously reported for one or more of the studied taxa. From a chemotaxonomical viewpoint, some compounds were found in all four Stachys members, evidencing a shared phytochemical relatedness. On the other hand, vicenin-2 and apigenin-glucoside were detected only in S. candida, while isoscutellarein-7-O-allopyranosyl-(1→2)-glucopyranoside was only detected in S. leucoglossa subsp. leucoglossa. Furthermore, S. candida and S. chrysantha had some compounds in common, evidencing (in phytochemical terms) their close affinity within the section Candida or the phyloclade Swainsoniana. Therefore, the data furnished herein may further contribute to ongoing phytochemical and chemotaxonomical investigations regarding the distribution of different compounds and categories thereof across different members and sections of the genus Stachys.