Isolation of phenolic constituents from Rhododendron yunnanense flowers as a potent cyclooxygenase-2 and vascular endothelial growth factor receptor-2 inhibitor: Phytochemical and molecular simulation studies

Mohamed Salaheldin Ahmed Abdelkader1*, Reda Ahmed Abdelhamid2, Mohamed Ezzat Abouelela2, Mostafa Ezzat Rateb3, Marwa Hassan Ahmed4 1Department of Pharmacognosy, Faculty of Pharmacy, Sohag University, Sohag, Egypt. 2Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut, Egypt. 3Microbial Natural Product Laboratory, School of Computing, Engineering & Physical Sciences, University of the West of Scotland, Paisley, UK. 4Department of Pharmacognosy, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt.


INTRODUCTION
Natural polyphenols are widely distributed phytochemicals in the plant kingdom that are considered a significant source for drug discovery and development (Asuzu et al., 2019). Recently, natural bioactive phenolics have gotten more attention as therapeutic agents due to their diverse bioactive functions, which potentially have beneficial implications in the underlying biological process in several diseases' regulation (Abhinand et al., 2020;Sayed et al., 2020). The treatment of cancer and chemopreventive and chemotherapeutic effects of polyphenolics, especially flavonoids, are intensively studied due to their low toxicity and antioxidant effects contributing to preventing and managing oxidative stress implicated mainly in cancer development (Asuzu et al., 2019).
The molecular targets for antineoplastic mechanisms include many pathways such as cell cycle arrest, apoptosis, necrosis, and angiogenesis inhibition (Jafari et al., 2014;Ko and Auyeung, 2013). Angiogenesis plays an essential role in increasing vasculature and blood supply needed for the growth, progression, and metastasis of tumors. Recent studies have shown that many isolated natural compounds exhibited antiangiogenic activity as a potential target for cancer treatments especially for solid tumors (Al Abd et al., 2017). It is well established that chronic inflammation is a risk factor for cancer development which is indicated by overexpression of inflammatory mediators such as cytokines, NF-κB, cyclooxygenase (COX), and vascular endothelial growth factor (VEGF) associated with various cancers development such as pancreatic, prostate, cervical, breast, lung, and colon cancers (Wong, 2019). Despite the fact that angiogenesis is regulated by multiple complex proangiogenic factors, the VEGF is a vital mediator of tumor angiogenesis in which its expression increases associated with tumor prognosis (Abhinand et al., 2020). Moreover, the expression of cyclooxygenase 2 (COX-2) by the tumor cells, particularly endothelial cells, was found to stimulate angiogenesis through the formation of prostaglandin E2 and interleukin-6 induction (Fosslien, 2000;Leahy et al., 2002). Recent studies suggest that COX-2 inhibitors could lead to a reduction of tumorigenesis by suppression of angiogenesis through the downregulation of VEGF production and other proangiogenic factors produced by tumor cells (Liu et al., 2000;Toomey et al., 2009).

General experimental procedures
1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded at 25°C with a Varian Inova 600 MHz NMR spectrometer. High-pressure liquid chromatography (HPLC) was carried out on Agilent 1260 Infinity preparative HPLC system with an Agilent Eclipse XDB-C18 column (5 m, 10 × 250 mm, Agilent Technologies, USA) monitored using an Agilent photodiode array detector. Detection was carried out at 220, 254, 280, 350, and 400 nm. All chemical reagents were purchased from Sigma-Aldrich and used without further purification. Medium-pressure liquid chromatography (MPLC) separations were carried out on Biotage system using Biotage reversed-phase and normal-phase silica prepacked columns. Detection was carried out at 220 and 280 nm. TLC was carried out on precoated TLC plates with silica gel 60 F254 (layer thickness 0.2 mm, Merck, Darmstadt, Germany).

Plant material
The flower of the plant was collected in May 2015 from the University of Aberdeen Botanical Garden, Aberdeen, UK. The plant was labeled by its name by the authority of the garden. The plant flowers were collected and dried under shade and finely powdered and the powder was used for the extraction procedure.

Molecular docking and simulations of binding activity
Molecular operating environment (MOE) program 2008.10 was used for the optimization of both examined ligands and receptors for docking studies. Molecular docking was carried out to investigate the affinity of isolated compounds to COX-2 (PDB ID: 3NL1) complexed with celecoxib and VEGFR-2 (PDB ID: 4ASD) complexed with sorafenib retrieved from the Protein Data Bank (http://www.rcsb.org/pdb) at the molecular level (Abdellatif et al., 2020;Bernstein et al., 1977). The downloaded proteins were prepared for docking by 3D protonation, deleting of unnecessary water molecules and all co-crystallized ligands and metals, and receptor fixation. Furthermore, the isolated compounds were subjected to 3D generation and energy minimization using Merck Molecular Forcefield (MMFF94s) to a gradient 0.05. The adopted docking procedure followed the standard protocol implemented in MOE 2008.10 and the geometry of the resulting complexes was studied using MOE's Pose Viewer utility. The interaction between the ligands and receptors binding site was generated and the results of docking were recorded as pose score (S) and binding energy.
Herein, we examined the isolated compounds (1-13) by the molecular docking technique to identify their potential as antiangiogenetic and anti-inflammatory. We utilized COX-2 (PDB ID: 3NL1) as representative proteins for inflammation and VEGFR-2 (PDB ID: 4ASD) for angiogenesis. The pose scoring, hydrogen bonding and interacting residues for these compounds with selected proteins were listed in Tables 1 and 2.
The analysis of docking results for the isolated compound with COX-2 receptor (Table 1, Fig. 2) revealed that all the tested compounds exhibited binding affinity to the receptor ranges from −20.4075 kcal/mol for compound 4 to −14.7821 kcal/ mol for compound 9, which was considered more active than the   (1) standard anti-inflammatory COX-2 inhibitor compound celecoxib (−13.1283 kcal/mol). Furthermore, the results of interactions with VEGFR-2 showed that compound 2 had the most stable binding energy to allosteric site of VEGFR-2 with a score of −17.6036 kcal/mol in comparison to sorafenib (−16.6507 kcal/mol) as the standard inhibitor to angiogenesis (Table 2, Fig. 2).
Furthermore, compound 2 revealed the highest binding affinity to VEGR-2 receptor site by interaction through hydrogen bonding and hydrophobic interactions with the amino acid residues from the receptor pocket (Fig. 4).
It noteworthy that the quercitrin (2) showed predominant activity for the two examined receptors [COX-2 (−19.4542 kcal/mol) and VEGFR-2 (−17.6036 kcal/mol)] (Fig.  5) more than the compared standards [celecoxib (−13.1283 kcal/mol) and sorafenib (−16.6507 kcal/mol)] which could be a lead compound for the development of dual acting compounds for the treatment of tumors either directly by antiangiogenic activity or by acting on inflammatory mediators as predisposing factors for tumor prognosis and development. The results are in agreement with previously reported activities of quercitrin (2) as an antioxidant, anti-inflammatory, anti-proliferative, antiangiogenic, and apoptotic inducer. These effects are directly linked to the anti-tumor property by its interference with receptors, cellular enzymes, and signal transduction systems (Ezzati et al., 2020;Stochmalová et al., 2013). The comparison between the interactions of compound 2 with the two receptors (Fig. 5) revealed that the hydroxyl group at 4′ position of ring B and the oxygen group in 3 position of flavonol together with oxygen groups of positions 1, 2, and 3 of the rhamnose moiety are essential for the interaction by hydrogen bonding for both receptors.