Chemical constituents isolated from the aerial parts of Helleborus cyclophyllus (A. Braun) Boiss. (Ranunculaceae), evaluation of their antioxidant and anti-inflammatory activity in vitro and virtual screening of molecular properties and bioactivity score

Abstract Chemical investigation of ethyl acetate extract from the aerial parts of Helleborus cyclophyllus (A.Braun) Boiss. led to the isolation of ten natural products, and their structures were identified to be: 2-deoxy-D-ribono-1,4-lactone (1), 2-O-feruloyl-L-malate (2), three flavonoids: quercetin 3-O-β-D-galactopyranoside (3), quercetin 3-O-6′′-(3-hydroxy-3-methyl-gloutaryl)-β-D-glucopyranoside (4) and quercetin 3-O-(2‴-caffeoylsophoroside) (5), 6-O-caffeoyl-1-O-p-coumaroyl-β-D-glucopyranoside (6), two ecdysteroids: 20-hydroxyecdysone (7) and polypodine B (8) and two bufadienolides: deglucohellebrin (9) and hellebrin (10), on the basis of MS and NMR spectra. This is the first report on the occurrence of compounds (2)-(6) in the genus Helleborus. The chemotaxonomic significance of these compounds was summarised. Bioactivity score, molecular and pharmacokinetic properties of the isolated compounds were calculated by online computer software program Molinspiration. Compounds showed promising bioactivity scores for drug targets. Moreover, some of the isolated phenolic compounds were tested for their antioxidant and antiinflammatory activities. Tested compounds presented antioxidant ability correlated to the presence of the phenolic hydroxyl groups. Graphical Abstract


Introduction
The small genus Helleborus of Ranunculaceae family (contains ca. 20 species) is widely distributed in Southeast Europe and West Asia. Previous phytochemical studies on Helleborus disclosed that steroids, including bufadienolides, phytoecdystones, and steroidal saponins were the main components (Meng et al. 2001;Cheng et al. 2014;Zhang et al. 2014;Iguchi et al. 2020 ] is the only species of this genus that is growing wild in Greece. H. cyclophyllus, locally known as 'Skarfi', is a perennial endemic of the Balkan Peninsula. Its leaves are complex, long, biconvex and toothed circularly forming a disk explaining the name 'cyclophyllus'. Flowering shoots bear 2-5 light green flowers, with flowering period March-May. Greek popular tradition attributes to 'Skarfi' many bizarre and magical qualities. The flowering period of this plant is late winter or early spring. It is certain, however, that even at the end of the 19th century, from Helleborus prepared drugs for the treatment of mania, melancholy, hypochondria, even epilepsy. Its use was in the form of powder or tincture (Strid and Tan 2002). Previous phytochemical investigation of the species reported the presence of palmitic/linoleic acids, phospholipids, b-sitosterol, uridine, small phenolic analogs, bufadienolides, furostanols, ecdysteroids and flavonoid derivatives (Philianos 1967;Philianos et al. 1983;Tsiftsoglou et al. 2018;Brillatz et al. 2020). Continuing our studies on this plant (Tsiftsoglou et al. 2018;Vartholomatos et al. 2020), we now report the investigation of the aerial parts of Helleborus cyclophyllus (A. Braun) Boiss.
Many studies on secondary metabolites of Helleborus species have been realised. Present study allowed the isolation and identification of three known quercetin glycosides that are reported for the first time in genus Helleborus. Compound (3) is a very abundant natural product and is isolated from various species. Some of these plants are Lonicera japonica Caprifoliaceae , Agrimonia pilosa Ledeb Rosaceae , Camptotheca acuminata Nyssaceae (Qun and Qiaoyu 2016), Laetia suaveolens Salicaceae (Estork et al. 2014), Houttuynia cordata Saururaceae , Kielmeyera variabilis Mart. & Zucc. Calophyllaceae (Cota et al. 2012). Till now, compound (4) is isolated only from two species Euphorbia bracteolata Euphorbiaceae (Liu et al. 2004) and Moringa oleifera Moringaceae (Kashiwada et al. 2012), but it is also detected in extracts of the plants Rosa spinosissima Rosaceae (Porter et al. 2012) and Oxytropis racemosa Fabaceae (Song et al. 2013). Compound (5) isolated from few species. Some of these plants are Petunia 'Mitchell' Solanaceae (Bloor et al. 1998) and Ranunculus peltatus subsp. peltatus Ranunculaceae ( b Gluchoff-Fiasson et al. 1997). Flavonoids have long time been considered as chemiotaxonomical markers of Ranunculaceae (Lebreton 1986) and in particular caffeoylated or feruloylated flavonol glycosides are typical of this family and of Helleborus spp. ( a Gluchoff-Fiasson et al. 1994). This is the first report of this compound in plant belong to the genus Helleborus.
As it concerns 2-O-feruloyl-L-malate (2) isolated for the first time from the plant Raphanus sativus Cruciferae (Nielsen et al. 1984). Till then, is reported as product after treatment with various reagents and cultivation under specific conditions of plants belong to Cruciferae family (Liang et al. 2006;Abdel-Farid et al. 2007;Pedras et al. 2008;Narv aez-Cuenca et al. 2012). This is the first research that reports its presence in plant of Ranunculaceae family.
According to our results, the two ecdysteroids polypodine B (8) and 20-hydroxyecdysone (7), and the two bufadienolides deglucohellebrin (9) and hellebrin (10) are presents in the underground (Tsiftsoglou et al. 2018), as well as in the aerial parts of Greek hellebore H. cyclophyllus. These compounds have also been detected in the water-soluble fraction of the methanol extract of the leaves of H. cyclophyllus collected from Epirus region (Greece) by other researchers (Yfanti et al. 2020).

In silico studies -drug likeness
Lipophilicity, as LogP values, is an important physicochemical property implicated in the biological response and pharmacokinetic behavior of bioactive agents. Hydrophilic/lipophilic nature of drug molecule affects drug absorption, bioavailability, drug receptor interaction, metabolism of molecule as well as toxicity. In order to evaluate the drug likeness of the isolated compounds we calculated various molecular properties e.g., partition coefficient (milogP), topological polar surface area (TPSA), hydrogen bond donors and acceptors, rotatable bonds, number of atoms, molecular weight considering the violations of Lipinski's rule of five. LogP values of all isolated compounds were found to be in the range of À2.38 to 1.60. This implies that all the examined compounds are hydrophilic because their log P is less than 5 and also that their permeability across cell membranes is high.
Low molecular weight drug molecules (less than 500) are easily transported, diffuse and absorbed compared to molecules with higher MW. Apart from compounds 3 and 4 all other compounds were found to have MW less than 500.
Total polar surface area (TPSA) is closely related to the hydrogen bonding potential of molecules. TPSA of the derivatives 1, 2, 7 and 8 was observed in the range 66.76-158.67 and is well below the 160 Å limit, indicating good oral bioavailability (Table S9). The upper limit for TPSA for a molecule to penetrate the brain is around 90 Å. Number of rotatable bonds is a topological parameter that measures molecular flexibility. Except for compound 1 all other compounds are 'flexible' because their rotatable bonds were found in the range 4-11 (Khan et al. 2013).
For in silico prediction, compounds with logBB value more than 0.3 are considered to have high absorption through BBB (Blood-Brain Barrier) whereas between 0.3 to À 0.1 and less than À0.1 is considered to be moderate and less absorbed through BBB. LogBB was measured by using the Rishton equation (Shityakov et al. 2013). None of the isolated compounds have result above the 0.3. Our findings do not support the permeability of these hybrids through BBB.