NMR Fingerprint Comparison of Cultivated Sideritis spp. from Cyprus

Medicinal and Aromatic Plants (MAPs) play an essential role in global health systems, since more than 80% of population use natural products in primary healthcare. Given that the global use of herbal medicines is exponentially increasing, as well as many MAPs products are introduced into the market, many cultivation practices are applied to produce high quality and standardized plant raw materials. Thus, the present study focuses on the chemical fingerprints of two cultivated Sideritis species. In Cyprus, Sideritis cypria Post and S. perfoliata L. subsp. perfoliata are widely used in traditional medicine. To date, there is no research work presenting the comparative chemical profiling between the aforesaid species using NMR methods. In this study, 1D and 2D NMR experiments were used to compare the chemical fingerprints of these species originated from conventional cultivation practices. Iridoids (ajugol, monomelittoside, and melittoside) and one flavone (4′-methyl-isoscutellarein 7-O-[6‴-O-acetyl]-β-D-allosyl(1→2)glucoside) were present in the infusion of S. perfoliata subsp. perfoliata. The phenylethanoid glycoside, acteoside, was detected in both samples. The phytochemical profiles of these cultivated species were similar to those of Sideritis species, indicating the positive impact of cultivation practices in MAPs.


Introduction
Medicinal and Aromatic Plants (MAPs) are sources of specialized products with therapeutic potential which could lead to the discovery of new drugs [1]. In last decade, the cultivation of MAPs is considered as a great aspect for developing high quality and standardized natural raw materials in adequate quantity for industrial scale production, as well as for avoiding their potential extinction.
As the cultivation of MAPs is continuously increasing, rapid and precise analytical methods for identifying their chemical fingerprint are essential need. Nuclear Magnetic Resonance (NMR) fingerprinting has enabled and accelerated analytical procedures and authentication process of plant extracts, since NMR method is a fast, simple, reproducible, and non-destructive technique [2].
In recent years, mountain tea (Sideritis spp., Lamiaceae), one of the most popular herbal preparations worldwide, is being sold as a high added value natural product in global markets. In Cyprus, the infusions of the aerial parts of S. cypria Post and S. perfoliata L. subsp. perfoliata are used in traditional medicine as agents for various ailments such as stomach discomforts, respiratory disorders, influenza, and common cold [3]. Previous studies on extracts and infusions of these species revealed their rich nutrition and phytochemical profiles, as well as their great in vitro biological effects, including antioxidant, anti-inflammatory, antimicrobial, and cytotoxic activities [4][5][6][7][8][9].
At present, there is no report on comparative metabolite profiling of S. cypria and S. perfoliata subsp. perfoliata, using NMR methods. Therefore, in this study, an analytical method based on 1D and 2D NMR spectra is proposed to compare the chemical fingerprints of these species, originated from conventional cultivation practices.

Plant Materials
Harvesting was performed from one-year-old plants cultivated at Athalassa farm (Nicosia, Cyprus) by the Department of Agriculture of Ministry of Agriculture, Rural Development and Environment of Cyprus (Sideritis cypria) in 2019 and at Akrotiri (Limassol, Cyprus) (Sideritis perfoliata subsp. perfoliata) in 2018. Plants of both species were produced from genetic material originated from the mother plantations of the National Department of Agriculture at Athalassa. Taxonomic identification of the species was verified by authors based on visual observations on mother plantations following the keys available to the online Flora of Cyprus-a dynamic check list [10]. All plants were at the flowering stage (June), while conventional cultivation practices were applied.

Preparation of the Infusions and NMR Spectroscopic Analysis
Precisely, 2 g of aerial parts of each plant were added in 100 mL boiled water for 5 min, separately [11]. Then, 2 mL of each sample were transferred in pre-weighed vials and were concentrated in a vacuum rotary evaporator at room temperature, yielding 0.5 g (S. cypria) and 0.4 g (S. perfoliata subsp. perfoliata). Distilled water was used as solvent for the infusions, being sure that any additive/impurity will not be included in the samples.
For NMR measurements, each sample was dissolved in 600 µL of CD 3 OD. 1D and 2D NMR spectra were recorded on Bruker 400 DRX instrument at 300 K. Chemical shifts are given in ppm (δ) and are referenced to the solvent signal at 3.31 ppm (CD 3 OD). COSY (COrrelation SpectroscopΥ) experiment was performed using standard Bruker microprograms. Parameters used to obtain (i) 1 H-NMR spectra: ns = 16, rg = 101 and acquisition time = 1 min and 30 s and (ii) COSY spectra: ns = 4, rg = 101 and acquisition time = 19 min and 30 s.

Results and Discussion
The two infusions of S. cypria and S. perfoliata subsp. perfoliata were prepared separately, following the monograph of EMA for Sideritis plants [11]. To obtain the first overview of the chemical classes of their constituents, the two samples were subjected to Thin Layer Chromatography (TLC) on silica gel with visualization under UV light (254 and 366 nm). In UV light at 366 nm, dark and blue spots appeared, possibly corresponding to flavonoids and phenolic derivatives. It is noteworthy to mention that terpenoids could not be easily detected in UV light, but after spraying with vanillin-sulfuric acid reagent and heating, they could be observed as purple spots. The TLC fingerprints of both samples after use of the aforesaid solution and heating revealed three major chemical classes: (i) terpenoids, (ii) flavonoids, and (iii) phenolic derivatives. Specifically, the infusion of S. cypria showed rich content in phenolic derivatives, whereas the infusion of S. perfoliata subsp. perfoliata seemed to contain additionally terpenoids.
As a next step, the 1 H-NMR spectra of the samples were obtained, unveiling the presence of different phytochemical classes of compounds. The comparative 1D-NMR fingerprints are presented in Figure 1. Based on the hydrogen peaks, the 1 H-NMR spectra of both infusions were divided into four main specific regions ( Figure 1). In the upfield region (δ 0.7-3.0 ppm) there appeared mainly signals of terpenoids. The second region (δ 3.1-4.9 ppm) included signals especially of sugars, whereas in the third region (δ 5.0-6.0 ppm) peaks of anomeric hydrogens of sugars and hydrogens of iridoids were illustrated. In the downfield region (δ 6.1-8.0 ppm) signals of flavonoids, phenylethanoid glycosides and phenolic acids were observed. Comparing the 1 H-NMR fingerprints, both spectra were similar in the first region ( Figure 2a). In the second region, three different peaks were spotted in the spectrum of the infusion of S. perfoliata subsp. perfoliata at δ 4.61 ppm (d, 7.5 Hz), 4.33 ppm (brs), and 4.19 ppm (d, 6.7 Hz) ( Figure 2b). Furthermore, some differences were detected in the last two regions between the samples (Figure 2c,d). Particularly, at the region among δ 5.0 to 6.0 ppm in the 1 H-NMR spectrum of the infusion of S. perfoliata subsp. perfoliata were observed hydrogen signals which could belong to iridoids and anomeric hydrogens of sugars, while in the spectrum of the infusion of S. cypria less peaks were shown at this region. Although the fingerprints of both samples were approximately similar at δ 6.1-8.0 ppm, some hydrogen signals at δ 6.15 ppm (dd, 1.9/6.1 Hz), 6.34 ppm (d, 6.6 Hz), 7.11 ppm (m), 7.32 ppm (d, 2.3 Hz), and 8.03 ppm (d, 8.3 Hz) were found only in the spectrum of the infusion of S. perfoliata subsp. perfoliata.
(a) In the upfield region (δ 0.7-3.0 ppm) there appeared mainly signals of terpenoids. The second region (δ 3.1-4.9 ppm) included signals especially of sugars, whereas in the third region (δ 5.0-6.0 ppm) peaks of anomeric hydrogens of sugars and hydrogens of iridoids were illustrated. In the downfield region (δ 6.1-8.0 ppm) signals of flavonoids, phenylethanoid glycosides and phenolic acids were observed. Comparing the 1 H-NMR fingerprints, both spectra were similar in the first region ( Figure 2a). In the second region, three different peaks were spotted in the spectrum of the infusion of S. perfoliata subsp. perfoliata at δ 4.61 ppm (d, 7.5 Hz), 4.33 ppm (brs), and 4.19 ppm (d, 6.7 Hz) ( Figure 2b). Furthermore, some differences were detected in the last two regions between the samples (Figure 2c,d). Particularly, at the region among δ 5.0 to 6.0 ppm in the 1 H-NMR spectrum of the infusion of S. perfoliata subsp. perfoliata were observed hydrogen signals which could belong to iridoids and anomeric hydrogens of sugars, while in the spectrum of the infusion of S. cypria less peaks were shown at this region. Although the fingerprints of both samples were approximately similar at δ 6.1-8.0 ppm, some hydrogen signals at δ 6.15 ppm (dd, 1.9/6.1 Hz), 6.34 ppm (d, 6.6 Hz), 7.11 ppm (m), 7.32 ppm (d, 2.3 Hz), and 8.03 ppm (d, 8.3 Hz) were found only in the spectrum of the infusion of S. perfoliata subsp. perfoliata. In the upfield region (δ 0.7-3.0 ppm) there appeared mainly signals of terpenoids. The second region (δ 3.1-4.9 ppm) included signals especially of sugars, whereas in the third region (δ 5.0-6.0 ppm) peaks of anomeric hydrogens of sugars and hydrogens of iridoids were illustrated. In the downfield region (δ 6.1-8.0 ppm) signals of flavonoids, phenylethanoid glycosides and phenolic acids were observed. Comparing the 1 H-NMR fingerprints, both spectra were similar in the first region (Figure 2a). In the second region, three different peaks were spotted in the spectrum of the infusion of S. perfoliata subsp. perfoliata at δ 4.61 ppm (d, 7.5 Hz), 4.33 ppm (brs), and 4.19 ppm (d, 6.7 Hz) ( Figure 2b). Furthermore, some differences were detected in the last two regions between the samples (Figure 2c,d). Particularly, at the region among δ 5.0 to 6.0 ppm in the 1 H-NMR spectrum of the infusion of S. perfoliata subsp. perfoliata were observed hydrogen signals which could belong to iridoids and anomeric hydrogens of sugars, while in the spectrum of the infusion of S. cypria less peaks were shown at this region. Although the fingerprints of both samples were approximately similar at δ 6.1-8.0 ppm, some hydrogen signals at δ 6.15 ppm (dd, 1.9/6.1 Hz), 6.34 ppm (d, 6.6 Hz), 7.11 ppm (m), 7.32 ppm (d, 2.3 Hz), and 8.03 ppm (d, 8.3 Hz) were found only in the spectrum of the infusion of S. perfoliata subsp. perfoliata.
(a) To get a better understanding about the observed differences of both samples, 2D 1 H-1 H-COSY spectra were carefully studied. The comparative spectra were presented in Figure 3, where the different correlation peaks were signed with green colour boxes. In the 1 H-1 H-COSY spectrum of the infusion of S. perfoliata subsp. perfoliata, we observed the major correlation peaks between hydrogens corresponding to iridoids, flavones and phenylethanoid glycosides. To be mentioned that the 1 H-1 H-COSY spectrum of the infusion of S. cypria revealed principally correlation peaks among hydrogens which belong to flavones and phenylethanoid glycosides. To get a better understanding about the observed differences of both samples, 2D 1 H-1 H-COSY spectra were carefully studied. The comparative spectra were presented in Figure 3, where the different correlation peaks were signed with green colour boxes. In the 1 H-1 H-COSY spectrum of the infusion of S. perfoliata subsp. perfoliata, we observed the major correlation peaks between hydrogens corresponding to iridoids, flavones and phenylethanoid glycosides. To be mentioned that the 1 H-1 H-COSY spectrum of the infusion of S. cypria revealed principally correlation peaks among hydrogens which belong to flavones and phenylethanoid glycosides. As an effort to interpretate the observed different signals in the 1D-/2D-NMR spectra of infusion of S. perfoliata subsp. perfoliata with those of S. cypria, we compared them with NMR spectra of previously isolated compounds from our works in Sideritis spp. [5,8,9,12,13]. Through the screening of the 1 H-NMR spectra of different compounds, we carefully noticed that the main different hydrogen signals in the spectrum of infusion of S. perfoliata subsp. perfoliata could be attributed to iridoids, namely ajugol [14] and monomelittoside and its 5-glucoside (melittoside) [15,16]. This assumption was also confirmed by the 2D 1 H-1 H-COSY spectra. The overlaid 1 H-NMR spectra of the infusion of S. perfoliata subsp. perfoliata and the three aforesaid iridoids are illustrated in Figure 4. In addition, we reported, herein, the hydrogen signals of these compounds and their corresponding signals in 1 H-NMR spectrum of the infusion (Table 1). Iridoids were not spotted in the 1 H-NMR spectrum of the infusion of S. cypria. Thus, we could assume that a principal difference among these infusions might be the absence of hydrogen signals belonging to iridoids in the infusion of S. cypria based on their NMR fingerprints. In Sideritis species, acetylated or not acetylated derivatives of hypolaetin, isoscutellarein, and their methylated derivatives are major constituents [11,17,18]. Isoscutellarein 7-O-allosyl(1→2)glucoside derivatives were also unveiled through this NMR process in both infusions. In the 1 H-NMR spectrum of the infusion of S. perfoliata subsp. perfoliata, a deshielded assignment at δ 8.03 ppm, d (8.3 Hz) was also observed which could be attributed to flavone structures with AB-type aromatic system in B ring. In addition, the hydrogen peaks at δ range 6.69-6.80 ppm could correspond to flavones. The 2D 1 H-1 H-COSY spectrum of this infusion demonstrated correlation peaks of this hydrogen with a hydrogen in the aromatic area at As an effort to interpretate the observed different signals in the 1D-/2D-NMR spectra of infusion of S. perfoliata subsp. perfoliata with those of S. cypria, we compared them with NMR spectra of previously isolated compounds from our works in Sideritis spp. [5,8,9,12,13]. Through the screening of the 1 H-NMR spectra of different compounds, we carefully noticed that the main different hydrogen signals in the spectrum of infusion of S. perfoliata subsp. perfoliata could be attributed to iridoids, namely ajugol [14] and monomelittoside and its 5-glucoside (melittoside) [15,16]. This assumption was also confirmed by the 2D 1 H-1 H-COSY spectra. The overlaid 1 H-NMR spectra of the infusion of S. perfoliata subsp. perfoliata and the three aforesaid iridoids are illustrated in Figure 4. In addition, we reported, herein, the hydrogen signals of these compounds and their corresponding signals in 1 H-NMR spectrum of the infusion (Table 1). Iridoids were not spotted in the 1 H-NMR spectrum of the infusion of S. cypria. Thus, we could assume that a principal difference among these infusions might be the absence of hydrogen signals belonging to iridoids in the infusion of S. cypria based on their NMR fingerprints. In Sideritis species, acetylated or not acetylated derivatives of hypolaetin, isoscutellarein, and their methylated derivatives are major constituents [11,17,18]. Isoscutellarein 7-O-allosyl(1→2)glucoside derivatives were also unveiled through this NMR process in both infusions. In the 1 H-NMR spectrum of the infusion of S. perfoliata subsp. perfoliata, a deshielded assignment at δ 8.03 ppm, d (8.3 Hz) was also observed which could be attributed to flavone structures with AB-type aromatic system in B ring. In addition, the hydrogen peaks at δ range 6.69-6.80 ppm could correspond to flavones. The 2D 1 H-1 H-COSY spectrum of this infusion demonstrated correlation peaks of this hydrogen with a hydrogen in the aromatic area at δ 7.12 ppm, d (8.3 Hz) (Figure 3), giving rise to the assumption that these hydrogens belong to flavones. Remarkably, the large deshielded shifts of these aromatic hydrogens indicated that this compound might be a 4 -methyl-isoscutellarein 7-O-allosyl(1→2)glucoside derivative. Given that some singlet hydrogen peaks were depicted at δ approximately 2.0 ppm, we could assume that the aforesaid compound could be acetylated. The overlaid 1 H-NMR spectra of Agronomy 2021, 11, 1503 6 of 9 the infusion of S. perfoliata subsp. perfoliata and the previously isolated compound from this species [5], 4 -methyl-isoscutellarein 7-O-[6 -O-acetyl]-β-D-allosyl(1→2)glucoside [19], are presented in Figure 5. Regarding the phenylethanoid glycosides, in the 1 H-NMR spectra of both samples we could observe the presence of characteristic hydrogen assignments of these compounds. However, it is not feasible to accurately identify the specific derivatives, as we cannot clearly detect the number and type of sugar moieties due to overlapped assignments in the δ region 3.1-5.5 ppm. Based on previous studies in genus Sideritis, the most common representative of this chemical class is acteoside which was identified in the 1 H-NMR spectra of both samples ( Figure 6) [11,17].
Consequently, the major differences between the infusions of S. cypria and S. perfoliata subsp. perfoliata were: (i) iridoids, which were detected only in the 1D-/2D-NMR spectra of S. perfoliata subsp. perfoliata and (ii) the acetylated flavone, 4 -methyl-isoscutellarein 7-O-[6 -O-acetyl]-β-D-allosyl(1→2)glucoside, which was found only in the latter species. However, both infusions presented assignments of flavones and phenylethanoid glycosides in the NMR spectra. These results are of great importance since both samples seemed to include bioactive constituents with various biological properties [17,18,20].
The presented NMR fingerprint method might be proposed as one of many potential tools for differentiation between the species.

Conclusions
The present study focused on the comparison of two cultivated Sideritis species from Cyprus, using an NMR process, revealing their similarities and differences. 1D-/2D-NMR spectra were applied for studying the chemical fingerprints of each infusion from S. cypria and S. perfoliata subsp. perfoliata. Both samples were rich in bioactive specialized products. However, the major differences between the samples were the presence of iridoids and one flavone in the infusion of S. perfoliata subsp. perfoliata. This work showed that the phytochemical profiles of these cultivated species were close to the typical profiles of Sideritis species, contributing to achieve a better insight to the potential impact of cultivation practices in MAPs.

Conclusions
The present study focused on the comparison of two cultivated Sideritis species from Cyprus, using an NMR process, revealing their similarities and differences. 1D-/2D-NMR spectra were applied for studying the chemical fingerprints of each infusion from S. cypria and S. perfoliata subsp. perfoliata. Both samples were rich in bioactive specialized products. However, the major differences between the samples were the presence of iridoids and one flavone in the infusion of S. perfoliata subsp. perfoliata. This work showed that the phytochemical profiles of these cultivated species were close to the typical profiles of Sideritis species, contributing to achieve a better insight to the potential impact of cultivation practices in MAPs.