Anti-Ageing Potential of S. euboea Heldr. Phenolics

In recent years, the use of Sideritis species as bioactive agents is increasing exponentially. The present study aimed to investigate the chemical constituents, as well as the anti-ageing potential of the cultivated Sideritis euboea Heldr. The chemical fingerprinting of the ethyl acetate residue of this plant was studied using 1D and 2D-NMR spectra. Isomeric compounds belonging to acylated flavone derivatives and phenylethanoid glycosides were detected in the early stage of the experimental process through 2D-NMR techniques. Overall, thirty-three known compounds were isolated and identified. Some of them are reported for the first time not only in S. euboea, but also in genus Sideritis L. The anti-ageing effect of the ethyl acetate residue and the isolated specialized products was assessed as anti-hyaluronidase activity. In silico docking simulation revealed the interactions of the isolated compounds with hyaluronidase. Furthermore, the in vitro study on the inhibition of hyaluronidase unveiled the potent inhibitory properties of ethyl acetate residue and apigenin 7-O-β-d-glucopyranoside. Though, the isomers of apigenin 7-O-p-coumaroyl-glucosides and also the 4′-methyl-hypolaetin 7-O-[6′′′-O-acetyl-β-d-allopyranosyl]-(1→2)-β-d-glucopyranoside exerted moderate hyaluronidase inhibition. This research represents the first study to report on the anti-hyaluronidase activity of Sideritis species, confirming its anti-inflammatory, cytotoxic and anti-ageing effects and its importance as an agent for cosmetic formulations as also anticancer potential.


Introduction
Natural Products (NPs) continue to be the major source for drug leads, providing unique structurally diverse chemical scaffolds [1]. In the chemistry of NPs, Nuclear Magnetic Resonance (NMR) turns out to be an indispensable tool in the process of screening crude plant extracts for bioactive metabolites [2]. However, dereplication and analysis of these extracts still remain a challenging research field for NMR spectroscopy. Plant extracts consist of unique and convoluted mixtures of bioactive NPs with complicated and unpredicted spectral patterns. Over the years, NMR experiments such as two-dimensional (2D-) NMR have enhanced and accelerated the costly and time-consuming analytical process of these extracts, developing new strategies in the design of drug candidates [3].
Sideritis euboea, a member of the genus Sideritis L. (Lamiaceae family), is an endemic species of Greece, occurring in Evia (Euboea) Island [4]. Traditionally, its infusion (known as mountain tea) is widely consumed against mild gastrointestinal discomfort and common flu [5][6][7][8]. Previous studies reported the rich chemical profile of its extracts, including various metabolites with significant pharmacological effects [4][5][6][7][8][9]. The constantly increasing demand for mountain tea results in serious concerns about the survival of the wild Sideritis populations due to their overharvesting. In parallel, the intensifying climate change combined with overharvesting is a particular threat to the biodiversity of these wild plants. Therefore, the cultivation of Sideritis species has been viewed as a dynamic solution that could contribute to reducing the extensive collection (or overharvesting) and the potential extinction of the natural wild populations, to produce raw materials in sufficient quantities for the industrial-scale production demanded, and most importantly, to affirm for high quality and standardized products.
As a continuous endeavor to explore and specify the complete phytochemical load of the cultivated species S. euboea, we present herein the isolation and identification of 33 chemical constituents from the ethyl acetate residue, including one fatty acid derivative, two diterpenes, three iridoids, twelve flavonoid derivatives, two lignans, six phenylethanoid glycosides and seven phenolic acid derivatives. To evaluate the anti-inflammatory activity of the specific residue of S. euboea, as well as its isolated secondary metabolites, an in vitro assay towards the inhibition of the enzyme Hyaluronidase (Hyal) was performed. Hyaluronidase is a glycosidase, whose main role is the degradation of hyaluronic acid, one of the major constituents of the extracellular matrix connective tissue and regulates the homeostasis and the humidity levels of the joints [10,11]. In humans, hyaluronidase can be found both in body fluids, such as sperm or blood, and in various organs, such as skin, eye, uterus, testis, or liver. Except for the degradation of hyaluronic acid, hyaluronidase has been found to take part in various inflammatory pathways, cancer metastasis, and many allergic reactions [12]. The inhibition of hyaluronidase is of great significance, mainly due to its vital role in skin early-ageing as well as for tumour development, as hyaluronidase can be considered as a tumour marker. As a result, skincare cosmetic products with high potency against the activity of hyaluronidase have been well developed. In addition, natural specialized products acting as hyaluronidase inhibitors are lead-compounds to the development of new anti-ageing products [13,14]. The anti-hyaluronidase potential of the isolated compounds was evaluated in silico and several positive hits were determined. The in silico results were further validated in vitro using a hyaluronidase assay and the recorded results were further rationalized based on their in silico determined interaction profile with hyaluronidase.
Regarding the isolation and the identification of the apigenin 7-O-p-coumaroyl-glucosides, 1D and 2D-NMR methods played a determinant role, enhancing the detection of each derivative and aiding in carefully identifying their assignments. In the NMR spectra of compounds 10 and 11, a distinct difference was observed at the signals of their glucose moieties. Specifically  (11) was observed at 4.98 (δc 71.5, HSQC). After fractionations, we succeeded to isolate pure compound 11, while the separation and isolation of its cis-isomer, compound 12, was not feasible. However, we tracked down the proton signals of these two isomeric acylated flavones, using 1 H-NMR, COSY, NOESY, HSQC and HMBC spectra. Regarding the NMR spectra of compounds 11 and 12, we assumed the following important observations for the p-coumaroyl group which could be used as characteristic markers; i) the olefinic protons of the cis-isomer are significantly shielded at δ 6.94, d 11.9 Hz (δc 146.0, HSQC) and δ 5.86, d 11.9 Hz (δc 115.8, HSQC) compared to the protons of the trans-isomer at δ 7.70, d 15.2 Hz (δc 146.5, HSQC) and δ 6.44, d 15.2 Hz (δc 115.0, HSQC) and ii) the aromatic protons of cis-isomer H-2 /6 were strongly deshielded at δ 7.71 (d, 8.7 Hz, H-2 /6 , δc 133.7 HSQC), while the protons H-3 /5 were found upfield at δ 6.77 (d, 8.7 Hz, δc 115.5 HSQC). It is important to stress out that we also observed the NOE signal between the olefinic protons of the cis-isomer in the NOESY spectrum, confirming the presence of the cis-derivative.
Comparing the 1 H-NMR profiling of apigenin 3 -/ 4 -p-coumaroyl-glucosides (10-12) to apigenin 6 -p-coumaroyl-glucoside (13), we detected two characteristic differences. Firstly, the methylene protons of glucose skeletons of compound 13 were strongly deshielded (δ 4.63, dd 11.9/2.3 Hz, H-6 a and 4.29, dd 11.9/8.3 Hz, H-6 b), as well as it was also observed a large upfield shift in the aromatic protons of aglycon and of p-coumaroyl moiety of the latter compound. According to literature, apigenin 4 and 6 -p-coumaroyl-glucosides were previously reported in several Sideritis species [24,45,48]. Of great interest is that the majority of these studies do not mention the isomer of apigenin 4 -p-coumaroyl-glucoside, whereas few of them describe the isolation of the trans-acylated flavone glucoside. To the best of our knowledge, the presence of apigenin 7-O-p-coumaroyl-glucosides (10-13) is mentioned for the first time in S. euboea. Though, apigenin 7-O-[3 -O-trans-p-coumaroyl]-β-D-glucopyranoside (10) is found for the second time in genus Sideritis [49]. The 1 H-NMR spectra of all the isolated apigenin derivatives are presented in Figure 1.
Lignans and neolignans consist of a large group of naturally occurring phenols in the Lamiaceae family. Nonetheless, these compounds are of rare occurrence in genus Sideritis.  [50]. Our research revealed the isolation and identification of two lignan analogues, a furofuran skeleton derivative namely pinoresinol-4-O-β-D-glucopyranoside (19) and a neolignan, known as (7S, 8R)-urolignoside (20). These specialized products are found for the first time in this species. Notably, this is the first report of (7S, 8R)-urolignoside in genus Sideritis. Phenylethanoid glycosides (PhGs) are among the major compounds found in genus Sideritis [47]. Several structures of this chemical category were thoroughly described in various Sideritis species, including mainly PhGs with two or three sugars. Compounds 21, 23 and 25 were previously found in S. euboea [6][7][8], while lamalboside (26) is reported in the specific species for the first time, herein. Furthermore, cis-acteoside (22) and cisleucosceptoside A (24) are new for the genus Sideritis. It is noteworthy to point out that compound 22 was isolated as a mixture with its isomer 21. The NMR techniques enabled us to obtain all the necessary information to identify these specialized products, even if the cis analogue was observed in a minor amount and only in one sample. As a first step, in the 1 H-NMR spectrum, we identified the proton signals of compound 21 and then, we noticed the rest proton peaks of compound 22. It was observed that the two isomers were mainly differentiated in two major points in 1D-/2D-NMR spectra; i) the proton signals of the olefinic protons of the caffeoyl moiety, i.e., for the cis-isomer. As a result, it is important to underlie that the large downfield of proton H-2 , as well as the upfield of protons H-7 and H-8 among with their multiplicity (d~11-12 Hz) in 1D-NMR might be used as characteristic markers for the presence of the cis-acteoside ( Figure 2). Moreover, our findings unveiled the appearance of one more additional minor cis-isomer specialized product, namely cis-leucosceptoside A (24). In 1D NMR fingerprinting, we discerned proton signals corresponding to the protons of a mixture of compounds 23 and 24 ( Figure 3). The latter isomers were differentiated in the proton signals of their feruloyl moieties in 1 H-NMR spectrum, i.e.,   Concerning the isolated phenolic derivatives, compounds 27 and 30-33 were reported in previous studies on various Sideritis species [49,51,52], whereas compounds 28 and 29 were not found before in the specific genus. However, the constituents 28-33 were isolated for the first time in S. euboea.
We should also point out that spotting the major chemical categories of the crude extract at the beginning of our workflow enabled us to confirm most of the isolated compounds and minimize potential artifacts during the analytical process.

In Silico Screening and In Vitro Evaluation of the Inhibition of S. euboea Ethyl Acetate Residue and Its Isolated Compounds towards Hyaluronidase
To explore the anti-hyaluronidase potential of the isolated compounds, we performed inverse virtual screening against hyaluronidase. The calculated free energy of binding of hyaluronidase against each isolated compound is presented in Figure 4. From the plot illustrated in Figure 4, it became evident that apigenin 7-O-β-D-glucopyranoside (compound 9) and its acylated derivatives (compounds 10-13  We then selected compounds 9, 12, 13, which showed enhanced in silico binding affinities to hyaluronidase, as well as compounds 17 and 18 which showed moderate binding affinity, to evaluate in vitro their anti-hyaluronidase activity. The five selected compounds (9, 12, 13, 17 and 18) as also the EtOAc residue of S. euboea were evaluated through an in vitro screening assay in two concentrations, 300 µg/mL and 500 µg/mL, in order to evaluate the inhibitory activity of each compound at these two concentrations ( Figure 5). The EtOAc crude extract of S. euboea showed weak inhibition at a concentration of 300 µg/mL, with a value of 18.55 ± 0.12%, whereas at 500 µg/mL, it showed moderate inhibition with a value of 35.67 ± 0.15%. However, compound 9, which is a derivative of apigenin glucoside illustrated moderate inhibitory activity (34.84 ± 0.08%) at a concentration of 300 µg/mL and very good (64.77 ± 0.06%) inhibitory activity at a concentration of 500 µg/mL. Compounds 12 and 13, which also consist of derivatives of apigenin glucoside showed weaker inhibition at 300 µg/mL, with values of 11.77 ± 0.06% and 0.8 ± 0.15%, respectively. However, at a concentration of 500 µg/mL compound 12 inhibited hyaluronidase with a value of 34.17 ± 0.08%. Between the two hypolaetin 7-O-allosylglucoside derivatives, which were studied for their anti-hyaluronidase potency, compound 17 showed weak inhibitory activity with 7.26 ± 0.11% at a concentration of 300 µg/mL, while at 500 µg/mL, the inhibition was moderate with a value of 35.67 ± 0.06%. Compound 18, however, showed weak inhibition at a concentration of 500 µg/mL, with a value of 10.96 ± 0.21% ( Figure 5). Additional studies were performed to determine the IC 50 values for the EtOAc residue as well as of the isolated compounds. For the EtOAc residue the IC 50 values were determined to 690.87 ± 2.01 µg/mL, while for compounds 9, 12 and 17 the IC 50 values were 927.15 ± 5.30 µM (400.90 ± 0.012 µg/mL), 1170 ± 8.72 µM (680 ± 0.015 µg/mL) and 978.01 ± 5.63 µM (650 ± 0.084 µg/mL) respectively, while for the compounds 13 and 18, the IC 50 values were found to be out of range.

In Silico Docking of Isolated Compounds towards Hyaluronidase
In order to further explore the identified differences in the in vitro activity of the different compounds against hyaluronidase, we analyzed the interaction profile of each compound which was selected via in silico docking, with hyaluronidase. Figure 6 and Figures S5-S8 of the Supplementary Materials illustrate the interactions recorded between the five isolated compounds with hyaluronidase. In Figure 6, it is shown the interaction profile of compound 9 (illustrated in light blue sticks) with hyaluronidase, where numerous hydrophobic, hydrogen bonds and pistacking interactions stabilize the formation of a complex. In this binding pose, the sugar moiety of compound 9 interacts with hyaluronidase through hydrophobic bonds with the active site residue Tyr202. Furthermore, the sugar moiety of compound 9 forms hydrogen bonds with the active site residues Glu131, Tyr202 and Asp292 ( Figure 6). The strong interaction is also affected through pi-stacking interactions between the flavonoid moiety (apigenin) of compound 9 and the enzyme through the active site aromatic residues Trp321 and Tyr75. In addition, the apigenin skeleton develops hydrophobic interactions with Tyr75, Tyr202, Tyr247, Tyr286 and Trp321. These data illustrate that this molecule strongly interacts specifically with the amino acid residues of the active site of hyaluronidase. These docking results could possibly explain the high inhibitory potency of compound 9 towards hyaluronidase. Compound 12 ( Figure S5, Supplementary Materials) forms hydrophobic interactions between its flavonoid moiety and hyaluronidase, through enzyme residues Tyr202, Tyr286, and Trp321, while additional hydrogen bonds are formed between apigenin and the residues Asn37 and Tyr286. Regarding its sugar moiety, it develops interactions with hyaluronidase through hydrogen bonds with Glu131. In addition, the coumaroyl moiety forms hydrogen bonds with Tyr202 as also hydrophobic interactions with Tyr202 and Tyr208. The coumaroyl moiety further forms hydrophobic interactions with Tyr202, Phe204 and Tyr208, while pi-stacking interactions are developed with Phe204. The calculated binding affinity of compound 12 for hyaluronidase was −10.5 kcal/mol. However, the respective inhibition at 300 µg/mL and 500 µg/mL is lower than the respective values of compound 9, as compound 9 interacts strongly with more active sited amino acid residues than compound 12.
The glucosidic analogue 13 ( Figure S6, Supplementary Materials) develops with hyaluronidase hydrophobic interactions between its coumaroyl moiety and the amino acids Tyr75, Tyr202, Tyr247, Tyr286 and Trp321. The binding of compound 13 with hyaluronidase is stabilized through pi-stacking interactions with the residues Tyr202, and Trp321 also via the coumaroyl moiety, leading to a binding affinity of −10.8 kcal/mol. Regarding the flavonoid moiety, it forms hydrophobic bonds with Tyr75 and Trp321. Lastly, the sugar moiety develops hydrophobic interactions with Tyr247 of hyaluronidase 47. Despite the fact that compound 13 has the most promising binding affinity comparing to the other isolated compounds, compound 9 displays the highest inhibitory potency towards hyaluronidase. This fact could be explained via in silico studies, as the coumaroyl moiety, in contrast to the sugar moiety of compound 13 interacts strongly with the active sited amino acid residues as it was analyzed above.
In contrast, compound 17 ( Figure S7, Supplementary Materials) showed a lower binding affinity with a value of −8.1 kcal/mol. In this case, the interaction takes place through hydrogen bonds between the sugar moiety and Arg134, and through hydrophobic bonds with Tyr202. Simultaneously, the flavonoid moiety forms hydrogen bonds with the amino acid residues Glu131, Tyr202, Asp292 and Tyr247. In addition, pi-stacking bond with Trp321 stabilizes the binding of compound 17 to hyaluronidase. The hypolaetin moiety of compound 17, also displays hydrophobic interactions with the active site amino acid residues Tyr75, Tyr202, Tyr247, and Trp321. In addition, the acetyl-β-D-allopyranosyl moiety, without the second sugar residue, of compound 17 interacts with hyaluronidase through hydrophobic bonds with the amino acid residues Phe212, Pro249 and Val251 which, though, do not belong to the active center. As illustrated in Figure 5, compound 17 displays lower inhibitory potency compared to compound 9. Compared to compound 12, although their inhibition values are in the same range, they differ in their binding affinities. This fact could be explained due to the formation of hydrophobic interactions between both the coumaroyl and the flavonoid moiety with the active site amino acid residues, whereas in compound 17 only the flavonoid moiety interacts strongly with the amino acids of the active center. Similar binding affinity showed compound 18 ( Figure S8, Supplementary Materials), with a value of −8.3 kcal/mol. However, compound 18 shows no inhibitory potency towards hyaluronidase. According to in silico docking results, compound 18 forms no hydrophobic interactions but displays hydrogen bonds with the residues Arg134 and Asp292 which are not part of the active center. This could deliver a potential explanation of the low inhibitory potential of this compound with respect to compound 9.

General Procedures
1D and 2D NMR spectra were recorded in CD 3 OD and CDCl 3 on Bruker DRX 400 instrument at 295 K. Chemical shifts are given in ppm (δ) and were referenced to the solvent signals at 3.31/49.0 and 7.24/77.0 ppm, respectively. COSY (COrrelation SpectroscopΥ), HSQC (Heteronuclear Single Quantum Correlation), HMBC (Heteronuclear Multiple Bond Correlation), and NOESY (Nuclear Overhauser Effect SpectroscopYy) (mixing time 950 ms) experiments were performed using standard Bruker microprograms. Column chromatography (CC): Sephadex LH-20 (Pharmacia) and Silica gel (Merck, Art. 9385, Darmstadt, Germany). Preparative-thin-layer chromatography (Prep-TLC) plates were pre-coated with silica gel (Merck, Art. 5721, Darmstadt, Germany). Fractionation was always monitored by TLC silica gel 60 F-254 (Merck, Art. 5554, Darmstadt, Germany) with visualization under UV (254 and 366 nm) and spraying with vanillin-sulfuric acid reagent. All obtained extracts, fractions, and isolated compounds were evaporated to dryness in a vacuum under low temperature and then were put in activated desiccators with P 2 O 5 until their weights had stabilized.

Plant Material
Aerial parts of S. euboea Heldr. were collected from a cultivated population (under organic farming conditions) in HAO DEMETER (Institute of Breeding and Plant Genetic Resources) in July 2017. The sample was authenticated by Dr. P. Chatzopoulou; a voucher specimen was deposited in the herbarium of the Aromatic and Medicinal Plant Department (code . The plant material was dried for 10 days at room temperature, and then powdered in a specific pulverization machine without freezing.

Extraction and Isolation
The whole process of the extraction of the air-dried powdered plant material (0.30 kg) was conducted as previously described [8]. The ethyl acetate residue (1.0 g) was fractionated by CC over silica gel (14.

Evaluation of the Inhibition of S. euboea Ethyl Acetate Residue and Its Isolated Compounds towards Hyaluronidase
The activity of hyaluronidase was determined through UV spectroscopy, by measuring the amount of N-acetylglucosaminoglycan, which is formed by the degradation of hyaluronic acid [53]. An amount of 400 U/mL of Hyalurorindase from bovine testes (Type I-S, lyophilized powder, 400-1000 units/mg, Sigma, Germany) was dissolved in 0.1 M acetate buffer, pH 3.5 and incubated with the aqueous solutions of the respective ligands at concentrations of 300 µg/mL and 500 µg/mL for 20 min at 37 • C. After the incubation of the mixture, 0.5 mg/mL BSA (Bovine Serum Albumin, lyophilized powder, crystallized ≥ 98%, Sigma, Germany) was added in each sample, and then water to reach a total volume of 200 µL. The total mixture was incubated in 37 • C for 60 min. The respective control samples had water in place of the samples. After the reaction time, 148 µL of each sample was transferred to an Eppendorf and 32.8 µL of boric acid was added. The total mixture was incubated at 100 • C for 5 min, to stop the reaction. The samples remained at room temperature (25 • C) and 820 µL of p-dimethylaminobenzaldeyde (DMAB) (cas No:100-10-7, Sigma, Germany) were added. The mixture was incubated at 37 • C for 20 min until the color of the producing N-acetylglucosaminoglycan was developed. The samples are measured at 590 nm by a UV spectrometer.

In Silico Docking of Isolated Compounds towards Hyaluronidase and in Silico Based Screening
For the ligand-based virtual screening, a 3D similarity search is conducted with the software WEGA [54] and the software SHAFTS [55]. For the docking calculations, we utilized the software Autodock Vina after setting a grid of 25 Å 3 centered in the defined binding site of every protein target. Protein targets and ligands are set for docking by means of AutoDock tools as in Forli et al. (2016) [56].
To validate the docking results we performed docking calculations of the well-known inhibitor of hyaluronidase liquiritigenin that its interaction with hyaluronidase has been reported [57]. For this, we used a different docking software (Maestro). Both software (Maestro and Autodock) resulted in the same interaction profile of liquiritigenin with hyaluronidase ( Figure S9, Supplementary Materials). Having determined the validity of our docking protocol, we performed docking calculations of compounds 9, 12, 13, 17 and 18 with Maestro software. The structures of the five selected compounds (9, 12, 13, 17 and 18) were built in the Maestro 10.2 utility, prepared with LigPrep, ionized at pH 7.0, minimized using the OPLS3 force field [58], while ConfGen in the thorough mode was used to generate possible bioactive conformations [59]. The crystal structure of hyaluronidase (PDB ID: 2PE4) was prepared using the Protein Preparation Wizard utility [60]. The receptor grid was generated centroid the catalytic site of the enzyme. The GlideXP algorithm was used for the docking of the ligands [61]. All the created poses were minimized post-docking ( Figure 6, Figures S5-S8, Supplementary Materials).

Conclusions
It is well known that plant extracts consist of a large variety of bioactive NPs. However, the chemical composition of the crude extracts is usually very complex. In the drug design process, profiling methods such as NMR techniques play an important role in their investigation. Using NMR methods from the early stages of a phytochemical analysis allows the rapid, non-destructive, reproducible, and costless detection of the major chemical groups of the specialized products, as well as the identification of the unknown or rare compounds.
The present study clearly showed the strength of NMR spectroscopy to identify chemical groups and spot isomeric compounds in plant mixtures, revealing the isolation and identification of many chemical compounds for the first time not only in the species S. euboea, but also in the genus Sideritis. These data are also of great chemophenetic interest. In vitro study on the inhibition of hyaluronidase by the EtOAc residue of S. euboea, as well as the isolated compounds, showed potent inhibitory properties of the residue and apigenin 7-O-β-D-glucopyranoside (compound 9) mainly, at a concentration of 500 µg/mL, while the rest apigenin 7-O-p-coumaroyl-glucosides and 4 -methyl-hypolaetin 7-O-[6 -Oacetyl-β-D-allopyranosyl]-(1→2)-β-D-glucopyranoside (compound 17) showed moderate inhibition at the same concentration. Furthermore, the in silico studies described the kind of interactions of the studied compounds with hyaluronidase. Based on the in silico virtual screening, compounds 9-13 showed similar binding affinity, however, their inhibition potential showed different values. In silico docking studies were performed in order to evaluate and explain the interaction profile of each selected compound.
Supplementary Materials: Figure S1: 1 H-NMR spectrum of EtOAc residue of cultivated S. euboea (CD 3 OD, 400 MHz), Figure S2: COSY spectrum of EtOAc residue of cultivated S. euboea (CD 3 OD, 400 MHz), Figure S3: HSQC spectrum of EtOAc residue of cultivated S. euboea (CD 3 OD, 400 MHz), Figure S4: HMBC spectrum of EtOAc residue of cultivated S. euboea (CD 3 OD, 400 MHz), Table S1: Chemical structures of the isolated compounds 1-33, Figure S5: Best pose of the interaction of compound 12 with hyaluronidase. The pi-stacking interactions are shown with light blue dashed lines and the hydrogen bonds with yellow dashed lines. Figure