South African Journal of Botany 121 (2019) 470–477 Contents lists available at ScienceDirect South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb Antioxidant activity and molecular docking study of Erythrina × neillii polyphenolics S.K. Gabr a,R.O.Bakra,⁎,E.S.Mostafaa,A.M.El-Fishawyb,T.S.El-Alfyb a Department of Pharmacognosy, Faculty of Pharmacy, October University for Modern Sciences and Arts, 11787 Giza, Egypt. b Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, 11562 Cairo, Egypt article info abstract Article history: Species of genus Erythrina have a great contribution in folk medicine; various species are utilized as a tranquilizer, Received 24 August 2018 to treat insomnia, inflammation and colic. Besides, Erythrina species have reported antioxidant, hepatoprotective Received in revised form 9 December 2018 and anxiolytic activities. Erythrina × neillii is a hybrid obtained through a cross between E. herbacea L. and Accepted 18 December 2018 E. humeana Spreng. It has not been well-studied for its chemical or biological profile; therefore it represents an Available online xxxx interesting field of study. In this study, seven phenolic compounds; two hydrolysable tannins (1,3), one phenolic fl – fi Edited by J Grúz acid (2) and four known avonoids (4 7) were isolated and characterized for the rst time in E × neillii and Erythrina genus except for vitexin (7). Isolated compounds were assessed for their antioxidant activities using Keywords: ORAC assay. 2″-O-galloyl orientin (6) exhibited the highest activity followed by 2″-O-galloyl vitexin (5). Flexible Erythrina × neillii molecular docking on heme oxygenase, an important stress protein that is involved in cellular protection, antiox- Polyphenolics idant and anti-inflammatory activities, justified the antioxidant activity of the isolated compounds. The best scor- ORAC ing was observed with 2″-O-galloyl orientin forming four binding interactions with residues, Arg 136 (two Docking interactions), Met34 and Gly139. Erythrina × neillii offered powerful and available antioxidant beside signifi- Heme oxygenase cantly active phytoconstituents. © 2018 SAAB. Published by Elsevier B.V. All rights reserved. 1. Introduction researches (Kapitulnik and Gonzalez, 2004; Pae et al., 2010; Son et al., 2013). Plant phenolics have beneficial effects against many pathological Oxidative stress (OS) is an imbalance between the reactive oxygen conditions including oxidative stress as they are able to scavenge free species (ROS) formation and the antioxidant defense mechanisms. At radicals (Brewer, 2011). their high concentrations, ROS can react with different macromolecules, Erythrina is a genus of flowering plants in the pea family (Fabaceae). therefore involved many disease processes. In our body, the cellular It contains more than 200 species distributed worldwide and known as antioxidant defense systems including glutathione (GSH), and ROS- “coral tree” (Neill, 1988). Traditionally, plants of this genus have a vari- scavenging enzymes, such as superoxide dismutase (SOD), catalase ety of uses such as tranquilizer, anti-inflammatory, in treatment of colic and glutathione peroxidase (GPX) regulate the levels of ROS (Valko and liver ailments (Chhabra et al., 1984; García-Mateos et al., 2001; et al., 2007). Ghosal et al., 1972). Erythrina is very rich in its phytoconstituents includ- Heme oxygenase-1 (HO-1) is a cellular stress protein that plays an ing alkaloids, flavonoids with its different classes, cinnamoylphenols, important role in the oxidative catabolism of heme leading to the stilbenoids, 3-phenoxychromones, coumastans, 3-phenyl-coumarins, formation of biliverdin (BV), free iron and carbon monoxide (CO). lignans, cinnamate esters, triterpenes, sesquiterpenes, long-chain car- Whereby, BV formed is rapidly converted to the strong antioxidant boxylic acids and long-chain alcohols (Callejon et al., 2014; Chacha bilirubin (BR), which is then converted back into BV through reacting et al., 2005; Majinda et al., 2005; Nkengfack et al., 2001; Pérez et al., with ROS allowing their neutralization. Therefore, HO-1 has its potential 2015; Wanjala and Majinda, 2000; Yenesew et al., 2003). A wide range ability to regulate oxidative and inflammatory which contribute to an of biological activities has been investigated including, antimicrobial ac- efficacy in controlling metabolic diseases and make it a target of several tivity with high efficacy against resistant organisms, anti-inflammatory, antidepressant, cytotoxic, hepatoprotective and muscle relaxant activi- ties (Anupama et al., 2012; Chacha et al., 2005; Majinda et al., 2005; ⁎ Corresponding author. Nkengfack et al., 2001; Setti-Perdigão et al., 2013). E-mail addresses: [email protected] (S.K. Gabr), [email protected] (R.O. Bakr), Erythrina × neillii Mabberley & Lorence is a hybrid derived from the [email protected] (E.S. Mostafa), elfi[email protected], ahlam.elfi[email protected] (A.M. El-Fishawy), [email protected] cross between E. herbacea and E. humeana. In continuation of our earlier (T.S. El-Alfy). studies on the pharmacognostical and genetic properties of this plant https://doi.org/10.1016/j.sajb.2018.12.011 0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved. S.K. Gabr et al. / South African Journal of Botany 121 (2019) 470–477 471 (Gabr et al., 2017), this study was undertaken to characterize the main 2.5. Quantitative estimation of phenolic and flavonoid contents active constituents in E × neillii leaf extract based on their spectroscopic data and comparison with reported literature. Those compounds were The total phenolic content of each fraction was determined by the tested for the in-vitro antioxidant activity using oxygen radical absor- Folin–Ciocalteau Reagent (FCR) using gallic acid as standard and bance capacity (ORAC) assay, moreover, flexible docking on HO-1 justi- expressed as milligram of gallic acid equivalents (GAE) per gram dry fied this activity. To the best of our knowledge, this is the firstreportfor extract (Sellappan et al., 2002). The absorbance was measured at λmax a chemical characterization of E × neillii and its activity. 765 nm using shimadzu UV–visible spectrophotometer (1800 UV- probe) after incubation for 2 h at room temperature. The total flavonoid 2. Material and methods content was determined by aluminum chloride colorimetric assay based on the quercetin calibration curve and expressed as milligram 2.1. General of quercetin equivalent per milligram dry extract (QE). The absorbance was measured at λmax 415 nm (Kosalec et al., 2004). Measurements Column chromatography (CC) was carried on, polyamide S-6 were carried out in triplicate. (E-Merk), Sephadex LH-20 (Pharmacia, Uppsala, Sweden), MCI gel column (CHP-20P, 75–150 μm; Mitsubishi Chemical Co., Dusseldorf, 2.6. Determination of oxygen radical absorbance capacity (ORAC) assay Germany). For paper chromatography, Whatman No. 1 and 3 sheets The evaluation of the oxygen radical absorbance activity by ORAC were used (E-Merk). Solvent systems (S1) BAW (Bu-HOAC-H2O, 4:1:5, upper layer), (S2) 15% HOAC. Detection of the developed was performed for the total extract and its fractions as well as the iso- chromatograms was performed under UV 254 and 365 nm light and ex- lated compounds using a 96-well microplate reader as previously reported (Huang et al., 2002; Nawwar et al., 2012). The antioxidant ca- posure to ammonia vapor. The NMR spectra were acquired in CD3OD on a Jeol ECA 500 MHz using tetramethylsilane (TMS) as the internal stan- pacity of the isolated compounds was measured by determining the fl fl ′ dard. UV spectrophotometer (Shimadzu UV/VIS-1601) was used for time course of the uorescence decay of uorescein, induced by 2,2 - analysis of pure samples in MeOH and in different UV shift reagents. azobis (2-amidinopropane) dihydrochloride (AAPH) compared with a ESI-MS was measured on spectrometer MAT 95 (Finigan-MAT, Bremen, vitamin E derivative, 6-hydroxy-2, 5, 7, 8-tetra-methylchroman-2- Germany). Oxygen radical absorbance capacity (ORAC) was performed carboxylic acid (Trolox), used as a positive control. on fluorometer, FLUO star OPTIMA, Franka Ganske, BMG LABTECH assisted by a Shimadzu UV–Visible-1601 spectrophotometer. For cyto- 2.7. Neutral red uptake (NRU) cytotoxicity assay toxic assay: Optical density was measured on a plate reader (Fluostar Omega, BMG Labtech, Offenburg, Germany). Human bladder carcinoma cells were sub-cultured twice a week and regularly tested for mycoplasma contamination. Cytotoxicity of the tested extracts was investigated using the neutral red uptake (NRU) 2.2. Reagents, chemicals and cells assay as described before (Repetto et al., 2008). The optical density was measured at 450 nm in a plate reader while all the experiments Fluorescein (Sigma–Aldrich), Etoposide (Alexis Biochemicals), were tested in duplicates. The IC values were obtained from the 6-hydroxy-2, 5, 7, 8-tetra-methylchroman-2-carboxylic acid Trolox 50 dose–response curves and expressed in mean ± SD. Etoposide was (Sigma–Aldrich), 2,2′-azobis (2-amidinopropane) dihydrochloride used as positive control. (AAPH) (Sigma–Aldrich). All solvents used in column chromatography were analytical grade. Human bladder carcinoma cell line 5637 was ob- 2.8. Extraction and isolation tained from CLS Cell Lines Service (Eppelheim, Germany). Cells were cultured in RPMI 1640 medium (BioWhittaker, Lonza, Verviers, The dried powdered leaves (1 kg) were defatted with petroleum Belgium) supplemented with 10% fetal