Dissertation committee:
Ελένη Σκαλτσά (Επιβλέπουσα), Καθηγήτρια, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Όλγα Τζάκου, Καθηγήτρια, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Αναστασία Καριώτη, Αναπληρώτρια Καθηγήτρια, Τμήμα Φαρμακευτικής, ΑΠΘ
Δέσποινα Περρέα, Ομότιμη Καθηγήτρια, Παθοβιοχημείας, Ιατρική Σχολή, ΕΚΠΑ
Διαμάντω Λάζαρη, Καθηγήτρια, Τμήμα Φαρμακευτικής, ΑΠΘ
Νεκτάριος Αληγιάννης, Αναπληρωτής Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Μιχαήλ Ράλλης, Αναπληρωτής Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Summary:
Background and Aim
Over the last decades, the research community has studied with interest plants and natural products as a basis for the design and development of new drugs for the treatment of various diseases, mainly cancer and cardiovascular disease, which are the two leading causes of death worldwide. Trying to reveal the great importance of the plants of our country with beneficial properties and high ethnopharmacological significance, the present study aims to: (a) isolate and identify the specialised products of Asplenium ceterach L. (rustyback) extracts, as well as investigate the effects of the traditional A. ceterach decoction in an experimental model of atherosclerosis in mice in order to highlight any beneficial properties for the cardiovascular system & (b) the bio-guided isolation and identification of the specialised products of Sideritis euboea Heldr. (mountain tea) for their cytotoxic activity, as well as for their possible inhibitory activity against hyaluronidase.
Methods
• A. ceterach L.
The plant material was successively extracted with cyclohexane, dichloromethane, and methanol. Part of the methanol extract was submitted to Charaux-Paris process, then was concentrated and the residue re-dissolved in boiling water. The water-soluble fraction was filtered and extracted with ethyl acetate and n-butanol successively. In addition, part of the cyclohexane extract was submitted to Gas Chromatography-Mass Spectroscopy (GC-MS) technique and to 1D- NMR (1 Dimensional- Nuclear Magnetic Resonance) experiment. The fractionations of the methanol extract, the ethyl acetate and n-butanol residues were done by various techniques such as MPLC (Medium Pressure Liquid Chromatography) VLC (Vacuum Liquid Chromatography), CC (Column Chromatography), TLC (Thin Layer Chromatography), HPLC (High Performance Liquid Chromatography). 1D- & 2D- NMR experiments were used for the structure elucidation of the isolated compounds.
The traditional decoction of A. ceterach was studied in an animal model of atherosclerosis for 8 weeks. 24 adult male mice (Mus musculus) SV129 wild type (wt) and 24 adult male mice PPAR-Alpha knock out (PPARa -/-) SV129 were randomly allocated into three groups per breed: Atheromatosis group (n=8), Atheromatosis + Rustyback group (n=8) and Control group (n=8). The two first groups received a high-fat diet for 8 weeks. Rustyback decoction was given orally as an aqueous solution in a daily dose. Body weight and serum biochemical parameters were determined at baseline, at 4 and 8 weeks. Moreover, at the baseline and at the end-point endocardiography analysis were performed. At the end of the study, the histopathological assessment was also carried out.
• S. euboea Heldr.
The plant material (originated from cultivated populations) was successively extracted with cyclohexane, dichloromethane and methanol. Extraction and fractionation methods were the same as described above. 1D- & 2D- NMR experiments were used for the structure elucidation of the isolated compounds.
The cytotoxic potential was evaluated, using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay on three human cancer cell lines DLD1, HeLa, and A549. The range of the examined concentrations were at 0-100 μM for 72h.
In addition, the ethyl acetate residue and its isolated compounds were evaluated in silico and in vitro for their inhibitory activity against hyaluronidase. The activity of hyaluronidase was determined through UV spectroscopy based on Morgan-Elson method. The examined in vitro concentrations were 300 μg/mL and 500 μg/mL.
Results
• A. ceterach L.
Overall, 14 natural compounds were isolated, belonging to flavonoids (compounds 1-4) and phenolic derivatives/ precursor metabolites (compounds 5-14). In the cyclohexane extract the presence of ethyl esters of palmitic, linoleic and α-linolenic acids was detected.
The results of the in vivo study show that: (a) the experimental model used for the development of atheromatosis is efficient since changes of their weight, as well as glucose, cholesterol, LDL cholesterol and triglycerides levels’ were observed in both mice types and (b) the endocardiography data provide evidence that the decoction of rustyback maintains stable the overall image of the mice when compared to the atheromatosis group and the baseline measurement.
• S. euboea Heldr.
In total, 48 natural products were isolated which were categorized into phytosterols (compounds 1-3), triterpenes (compound 4), diterpenoids (compounds 5-8), iridoids (compounds 9-12), flavonoids (compounds 13-27), lignans/neolignans (compounds 28 & 29), phenylethanoid glycosides (compounds 30-37), phenolic acids (compounds 38-46) and lipid acid derivatives (compounds 47 & 48).
The IC50 values of siderol in DLD1, HeLa, and A549 cells were 26.4 ± 3.7 μΜ (DLD1), 44.7 ± 7.2 μΜ (HeLa) and 46.0 ± 4.9 μΜ (A549). The diterpenoid showed an enhanced cytotoxic effect on the DLD1 cell line compared to HeLa and A549 cells, where siderol’s IC50 values were almost two-fold the IC50 value in the DLD1.
The compound apigenin-7-Ο-β-D-glucopyranoside and its acylated derivatives showed the most optimum binding profile in silico. Compounds apigenin 7-O-β-D-glucopyranoside, apigenin-7-O-[4''-O-Ζ-p-coumaroyl]-β-D-glucopyranoside, apigenin 7-O-[6''-O-Ε-p- coumaroyl]-β-D-glucopyranoside demonstrated enhanced in silico binding affinities to hyaluronidase, as well as compounds 4'-methyl-hypolaetin 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)-6''-O-acetyl-β-D-glucopyranoside (moderate binding affinity in silico) and the ethyl acetate residue were evaluated in vitro for their anti-hyaluronidase activity. 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%. The apigenin 7-Ο-β-D-glucopyranoside, exhibited moderate inhibition (34.84 ± 0.08%) at a concentration of 300 μg/mL and very good inhibitory activity (64.77 ± 0.06%) at a concentration of 500 μg/mL. Apigenin 7-O-[4''-O-Ζ-p-coumaroyl]-β-D-glucopyranoside and apigenin 7-O-[6''-O-Ε-p-coumaroyl]-β-D-glucopyranoside showed weaker activity at a concentration of 300 μg/mL (11.77 ± 0.06% and 0.8 ± 0.15%, respectively). However, at a concentration of 500 μg/mL apigenin 7-O-[4''-O-Ζ-p-coumaroyl]-β-D-glucopyranoside 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, 4'-methyl-hypolaetin 7-O-[6'''-O-acetyl-β-D-allopyranosyl]-(1→2)-β-D-glucopyranoside showed weak inhibitory activity (7.26 ± 0.11%) at concentration of 300 μg/mL, whereas at concentration of 500 μg/mL, the inhibition was moderate with a value of 35.67 ± 0.06%. On the contrary, 4'-methyl-hypolaetin 7-O-[6'''-O-acetyl-β-D-allopyranosyl]-(1→2)-6''-O-acetyl-β-D-glucopyranoside showed weak inhibitory activity at the same concentration (500 μg/mL, 10.96 ± 0.21%).
Conclusions
The present study reveals the importance of two plants of Greek Flora (A. ceterach L. & S. euboea Heldr.) with high ethnopharmacological significance.
Regarding the phytochemical investigation of the two plants:
14 natural products were isolated of which 10 are isolated for the first time from A. ceterach L. and 6 of them are mentioned for the first time in genus Asplenium L.
48 natural products were isolated of which 33 are isolated for the first time from S. euboea Heldr and 10 are mentioned for the first time in genus Sideritis L.
As a result, this study importantly contributes to the chemotaxonomy of genus Asplenium L. and Sideritis L.
Given that the plant material of S. euboea was originated from cultivated populations, it should be mentioned that this study indicates that the cultivation for this species is feasible, providing plants with rich phytochemical profiles.
Regarding the in vivo study of the decoction of A. ceterach, the present study is the first report on the investigation of this traditional decoction in animal model of atheromatosis (mice), examining a potential mode of action on the lipid metabolism, glucose and inflammation control through PPARa receptors. Specifically, the results provide evidence that the traditional decoction might play a protective role in the progress of the disease by stabilising or delaying its development. However, further studies are essential to clarify the mechanism of action, dosage and possible side effects. Importantly, this study confirms the use of this plant as an anti-inflammatory and diuretic agent in folk medicine.
Regarding the in vitro studies of S. euboea, the present study consists the first report on the cytotoxic activity of siderol which is one of the major compounds of genus Sideritis L., unveiling its beneficial potential as a remarkable cytotoxic agent. Furthermore, the in silico and in vitro studies revealed the potent inhibitory properties of the ethyl acetate residue of S. euboea and its isolated compounds. This research represents the first study on the antihyaluronidase 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. In the end, the aforementioned results confirm the traditional uses of genus Sideritis L. as an anti-inflammatory agent.
