Antioxidant Activity and Molecular Docking Study of Erythrina × Neillii Polyphenolics

South African Journal of Botany 121 (2019) 470–477

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South African Journal of Botany

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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 activities (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

(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 bovine serum (Sigma–Aldrich, ether then subjected to extensive extraction using 70% methanol Taufkirchen, Germany) and antibiotics (100 U/ml penicillin, 100 μg/ml under reflux (5 L × 5, 60 °C) till exhaustion. The defatted methanolic streptomycin; Sigma Aldrich, Taufkirchen, Germany) at 95% humidity, extract was concentrated under reduced pressure to yield a viscous res- 5% CO2 and 37 °C. idue (200 g). The residue was precipitated from H2O using excess methanol (1:10) followed by evaporation of the filtrate under vacuum to 2.3. Plant material afford 150 g. The dried extract was subjected to CC using polyamide

(500 g, 100 × 5 cm) beginning with H2O then decreasing the polarity Leaves of Erythrina × neillii Mabberley & Lorence were collected dur- gradually using MeOH from 100% H2O to 100% MeOH) to afford 50 fl ing the owering stage, October 2012 from El-Zohria garden, Cairo and fractions (500 ml each), which were collected into 6 collective fractions fi were kindly identi ed by Dr. Gwilym P. Lewis, Legume Research Leader, (I–VI) based on paper chromatographic investigation assisted by UV- Comparative Plant & Fungal Biology, Royal Botanic Gardens, Kew, UK. A light and spray reagents (Marzouk et al., 2009). Fraction I was devoid voucher specimen was deposited in the Herbarium of the Faculty of of polyphenolic compounds. Compound (1) (12 mg) was obtained Pharmacy, MSA University under registration number (RS020) and at from fraction II (2.05 g) eluted with 10% methanol and further purified the Department of Pharmacognosy, Faculty of Pharmacy, Cairo Univer- on sephadex LH-20 and water saturated n-BuOH as eluent. Compound sity (no.1-11-2012). (2) (20 mg) was isolated from fraction III (6.68 g) eluted with 20% MeOH that was further fractionated on a MCI gel column and eluted 2.4. Extraction and fractionation of extracts for quantitative estimation, with water saturated n-BuOH to afford two individual sub-fractions, ORAC and cytotoxic assay whereas compound (2) was isolated from sub-fraction (ii). Compound (3), (22 mg) was isolated from fraction IV (5.41 g), eluted with 30% Two hundred grams of powdered leaves were extracted with 70% MeOH and purified over Sephadex LH-20 using 50% MeOH. Fraction V methanol then fractionated beginning with Pet. ether (40–60 °C) (2.78 g) eluted with 40% MeOH was isolated using preparative PC and followed by methylene chloride, ethyl acetate and butanol. The extrac- S1 then finally purified on a sephadex LH-20 eluted with 50% MeOH tion with each solvent continued till exhaustion. Each extract was to yield compound (4) (15 mg). The repeated preparative PC of fraction distilled off under reduced pressure and dried to constant weight then VI (3.2 g, eluted with 70% MeOH), and S1 as an eluent afforded com- kept for determination of the total phenolic and flavonoid contents as pounds 5 (20 mg), 6 (15 mg) and 7 (10 mg), each was individually pu- well as the antioxidant and cytotoxic activities. rified on Sephadex LH-20 column using 50% aqueous MeOH. 472 S.K. Gabr et al. / South African Journal of Botany 121 (2019) 470–477

2.9. Isolated compounds Table 1 1 H-NMR spectral data (500 MHz, CD3OD) of the Compounds 4–7 in ppm from TMS, mul- tiplicities and J values (Hz) are given in parentheses. 2,3-O-hexahydroxydiphenoyl-(α/β)-glucopyranose (1):off-white amorphous powder; UV: λmax(MeOH): 259. M, 482, ESI-MS (negative 4567 − - − mode) m/z 481 [M-H] , 301 [M-H-180] , 300 [ellagic acid-H] ,275 Aglycone 1 and 249. H-NMR: Glucose moiety in α-anomers: δ ppm 5.25 (1H, d, H-3 6.09 6.52 6.57 J = 3.5 Hz, α-H-1), 4.49 (1H, dd, J = 3.5 Hz, α-H-2), 3.71–4.56 (m, H- H-6 6.3 (d; 2) 6.08 6.09 6.51 –– – 4-H-6α). Glucose moiety in β-anomers: 5.11 (1H, d, J = 8 Hz, β-H-1), H-8 6.35 (d; 2) ′ β β H-2 8.07 (d; 8) 8.11 (d;8) 7.45 (d; 2) 8.12 (d; 7.5) 4.69 (1H, dd,J=8and7.5, -H-2), 4.84 (1H, t, J = 7.5 Hz, -H-3), H-3′ 6.96 (dd; 2,8) 6.93 (d;8) – 6.82 (d; 7.5) 3.71–4.56 (m, H-4-H-6β). Hexahydroxy-diphenoyl moiety in α-&β- H-5′ 6.96 (dd; 2,8) 6.93 (d; 8) 6.89 (d; 8) 6.82 (d; 7.5) anomers: 6.35 (2H in total, s, H-3'α/β HHDP), 6.45 (1H, s, H-3″α/β H-6′ 8.07 (d; 8) 8.07 (d; 8) 8.11 (d; 8) 7.56 (dd; 8, 2) 8.12 (d; 7.5) 13 α β α HHDP). C-NMRglucose moiety: 61.04 (C-6 / ), 67.40 (C-4 ), Glucosyl 67.53 (C-4 β), 72.08 (C-2 α), 74.41 (C-2 β), 77.42 (C-3 α), 77.43 (C-5 H-1″ 4.92 (d, 7) 4.97 (d;8) 4.97 (d; 8) 4.99 (d; 9.6) α/β), 80.08 (C-3 β), 90.17 (C-1 α), 93.48 (C-1 β). Hexahydroxy- H-2″ 5.50 (t;9.3) 5.52 (t; 9.5) diphenoyl moiety in α-&β-anomers: 105.20, 105.53 (C-3′ &C-3″), H-3″ 3.71 (t;9.3) 3.71 (t; 9.1) H-4″ 113.92, 114.16 (C-1′ &C-1″), 125.25, 125.56 (C-2′ &C-2″), 135.25, H-5″ ′ ″ ′ ″ 135.38 (C-5 & C-5 ),144.63, 144.70, 145.10, 145.29 (C-4 , C-4 and C- H-6″ 4.61 (Br.d; 10) 6′ &C-6″), 169.09, 169.52 (C=O). Galloyl Brevifolin carboxylic acid (2):Off-white amorphous powder; UV: H-2″′,6″′ 6.74 (s) 6.74 (s) λ max (MeOH) 278, 350, 362; M, 292, ESI-MS (negative mode) m/z 291 Coumaroyl − 1 [M-H] , 247 [M–H-CO2]. H-NMR: δ ppm 2.51 (2H, s, H-3 and H-3′); 2″′,6″′ 7.47 (d;8) 3.75 (1H, dd, J = 4 and 2 Hz, H-2); 6.95 (1H, s,H-7).13C-NMR: δ ppm 3″′,5″′ 6.81 (d;8) ″′ 39.36 (C-3); 40.32 (C-2); 109.19 (C-7); 121.21 (C-4b); 121.21 (C-6a); 7 7.81 (d; 15) 8″′ 6.45 (d; 15) 138.58 (C-9); 138.58 (C-10a); 141.30 (C-8); 142.28 (C-10); 145.96 (C-4a); 161.02 (C-6); 168.01 (C-1); 195.13(C-4). α β 4 2,3-digalloyl-( / )- C1-glucopyranose (nilocitin) (3):Off-white (PDB code: 1N3U) was downloaded from the Protein Data Bank λ amorphous powder; UV: max (MeOH) 274; M, 484, ESI-MS (negative (www.pdb.org). The predicted binding energies for the different com- − – − 1 mode) m/z 483 [M-H] ,331[M H-galloyl] , 169 and 125. H-NMR: pounds as well as the binding interactions were determined and δ – α β α β glucose moiety: ppm 3.6 3.95 (m,H-4 and H-4 ,6-H and 6-H ), compared. 4.0–4.21 (m, 5-Hβ and 5-Hα), 4.94 (1H, d, J = 9.5 Hz, H-1β), 4.98 (1H, dd, J = 9.5 & 3.5 Hz, H-2α), 5.10 (1H, d, J = 3.5 Hz, H-1α), 5.22 (1H, dd, J = 9.5 and 3.5 Hz, H-2β), 5.43 (1H, t, J = 9.5 Hz, H-3α), 5.52 Table 2 13 – (1H, dd, J = 9.5 and 9 Hz, H-3β). Galloyl moieties in α-and C-NMR spectral data (125 MHz; CD3OD) of compounds 4 7. β-anomers: δ ppm 6.87(2H, s, H-2′,6′), 6.88 (2H, s,H-2″, 4567 ″ 13 α δ 6 ). C-NMR: -glucose moiety: ppm 60.6 (C-6), 65.13 (C-4), 73.20 C-2 157.04 166.60 166.60 164.00 (C-2, 3 and 5), 89.79, 94.85 (C-1 α/β). β-glucose moiety: δ ppm 60.58 C-3 134.60 102.70 102.70 102.49 (C-6), 68.79 (C-5 α/β), 68.82 (C-4),73.83 (C-2), 75.50 (C-3), 94.50 C-4 178.19 182.99 182.10 183.00 (C-1). Galloyl moieties in α-andβ-anomers: δ ppm 109.29 C-5 160.40 161.70 161.70 161.00 C-6 98.92 98.99 98.10 97.58 (C-2′,6'α/β,2″,6″ α/β), 119.02120.64, 121.35 (C-1′ α/β,1″ α/β), 138.67, C-7 165.00 163.30 163.30 157.60 139 (C-4'α/β,4″α/β), 145.66 (C-3′,5′ α/β,3″,5″ α/β), 165, 165.2, 165.4, C-8 92.81 104.60 104.61 104.67 166 (C=O α/β,C′ =Oα/β). C-9 157.04 157.00 157.03 156.05 Kaempferol-3-O-(6″-p-coumaroyl-β-glucopyranoside) (4): C-10 105.16 102.70 102.70 102.98 ′ Brown amorphous powder; UV: λ (MeOH): 266, 369; (NaOMe) C-1 121.34 122.00 122.00 122.00 max C-2′ 130.91 129.10 113.70 128.68 273, 325, 398; (NaOAc) 271, 302 (shoulder), 356; (NaOAc/H3BO3) C-3′ 115.88 115.90 145.90 115.98 270, 365; (AlCl3) 270, 345, 405; (AlCl3/HCl) 270, 342, 403.M,594,ESI- C-4′ 160 161.70 150.00 161.18 − MS (negative mode) m/z 593 [M-H] , 323, 285, and 119.1H-NMR and C-5′ 115.88 115.90 115.70 115.98 13C-NMR (Tables 1 and 2). C-6′ 130.91 129.10 120.00 128.68

2″ O-galloyl vitexin (5): yellow amorphous powder. UV: λmax Glucosyl (MeOH): 273, 333; (NaOMe) 283, 302 (shoulder), 386; (NaOAc) 283, C-1″ 97.84 72.00 72.00 73.48 C-2″ 72.02 73.00 73.01 70.93 392; (NaOAc/H3BO3) 275, 334; (AlCl3) 280, 306, 349, 386; (AlCl3/HCl) − C-3″ 77.25 77.00 76.89 77.73 280, 305, 344. ESI-MS (negative mode) m/z 583 [M-H] , 431 [M-H- C-4″ 70.80 71.50 7.40 71.50 − 1 13 galloyl] ,169. H-NMR and C-NMR (Tables 1 and 2). C-5″ 75.50 82.20 82.20 81.43 2″ O-galloyl orientin (6): Yellow amorphous powder, ESI-MS (neg- C-6″ 62.50 61.80 61.80 61.38 ative mode) m/z 599 [M-H]−, 429 [M-H-gallic acid]−, 447 [M-H- Galloyl − 1 13 galloyl] ,169. H-NMR and C-NMR (Tables 1 and 2). C-1″′ 120.00 120.00 Vitexin (7): UV: λmax(MeOH): 273, 333; (NaOMe) 275, 300 (shoul- C-2″′, C-6″′ 109.20 109.20 C-3″′, C-5″′ 145.20 145.20 der), 340; (NaOAc) 276, 352; (NaOAc/H3BO3) 275, 334; (AlCl3)280, ″′ 306, 349, 386; (AlCl /HCl) 280, 306, 349, 386. M,432,ESI-MS(negative C-4 136.68 136.98 3 C-7″′ 167.23 167.70 mode) m/z 431 [M-H]−1H-NMR and 13C-NMR (Tables 1 and 2). Coumaroyl ″′ 2.10. Molecular docking C-1 125.94 C-2″′, C-6″′ 130.91 C-3″′, C-5″′ 115.57 Flexible molecular docking study was carried out on Heme oxygen- C-4″′ 160.00 ase for the isolated compounds from the aqueous methanolic extract of C-7″′ 145.34 E × neillii using Molecular Operating Environment (MOE) software C-8″′ 115.81 C=O 166.32 (MOE_2014.0901). The X-ray crystal structure of Heme oxygenase S.K. Gabr et al. / South African Journal of Botany 121 (2019) 470–477 473

3. Results and discussion glucose protons, respectively (Nawwar et al., 1984). A pair of doublet

of doublet at δH ppm 4.49 (1H, dd, J = 3.5, 8 Hz, α-H-2) and at 4.69 Erythrina × neillii methanolic extract was fractionated with petro- (1H, dd, J = 3.5, 8 Hz, β-H-2), is assignable to the H-2 glucose proton, leum ether (PE), methylene chloride (MC), ethyl acetate (EA) and buta- in both α- and β- anomers, respectively. It revealed a resonance at δH nol (Bu) till exhaustion. The resultant pooled extracts were separately ppm 4.84 (1H, t, J = 7.5 Hz), assignable to the H-3 glucose protons. concentrated under reduced pressure and kept for determination of The downfield H-2 and H-3 sugar resonances beside that of C2 and C3 the phenolic and flavonoid contents beside the antioxidant and cyto- confirmed their attachment to HHDP. Therefore assigning compound toxic activities. The phenolic content was estimated as gallic acid equiv- (1)tobe2,3-O-hexahydroxydiphenoyl-(α/β)-glucopyranose alent (GAE) (y = 0.0111× −0.0339, R2 = 0.9901). The total methanolic (Tanaka et al., 1986). extract showed the highest content (10.12 mg/g) followed by the EA Brevifolin carboxylic acid(2) was obtained as an off-white amor- (9.56 mg/g) then Bu fraction (9.34 mg/g), while the lowest concentra- phous powder with a pseudomolecular ion peak [M-H]− at m/z 291 tion was observed with MC extract (9.1 mg/g). Whereas, the flavonoid with fragment at m/z 247 [M-H-44]− denoting the loss of carboxylic content was estimated based on the complex formed with aluminum group.1Hand13C-NMR analysis were in agreement with the literature chloride and quantified as quercetin equivalent (QE) (y = 0.0047×, for brevifolin carboxylic acid (Nawwar et al., 1994). 2 4 R = 0.9637). Bu showed the highest flavonoid content (15.9 mg/g 2,3 digalloyl-(α/β)- C1-glucopyranose (nilocitin)(3), appeared as QE) followed by the EA (13.5 mg/g QE) then the total methanolic extract an off-white amorphous powder with a pseudomolecular ion peak (12.07 mg/g QE), while the MC fraction (7.34 mg/g) showed the lowest [M-H]− at m/z 483. Fragment at m/z 331 denotes the loss of a galloyl concentration. moiety [M-H-152]− which was further confirmed by fragments at m/z ORAC assay helped in determination of the antioxidant activity 169 and 125 which are diagnostic of gallic acid moiety. 1H-NMR spec- where, the fluorescence decay was detected and the % inhibition of trum showed the characteristic two proton singlets of the two galloyl the decay was calculated (Table 3). The antioxidant activity of all ex- group at δH ppm 6.88 and 6.87 (2H, s, H-2″,6″) in the aromatic region tracts increased in a dose-dependent manner where, EA fraction while in the aliphatic region, two different patterns of proton signals be- showed the highest activity followed by Bu, which may be attributed longing to α/β anomeric mixture of disubstituted glucose appeared. A to the high concentration of phenolic contents. While the PE and MC pair of doublets centered at δH ppm 5.10 (1H, d, J =3.5Hz,H-1α)and fractions showed the lowest activity. 4.94 (1H, d, J = 9.5, H-1β) assigned the α and β anomeric glucose pro- The potential cytotoxicity of the E × neillii total methanolic extract tons indicating a free anomeric OH group. The downfield glucose pro- and its fractions was evaluated on human bladder carcinoma cell line tons at δH ppm 4.98 (1H, dd, J = 9.5 and 3.5 Hz, H-2α) and 5.22 (1H, 5637 using Neutral Red Uptake (NRU) assay (Repetto et al., 2008). Bu dd, J = 9.5 and 3.5, H-2β) assignable to H-2 glucose protons, in addition showed the highest cytotoxicity (IC50 of 40.92 μg/ml) followed by EA to the downfield shift at δH ppm 5.522 (1H, dd, J = 9.5 and 9 Hz, H-3β) (IC50 of 50.09 μg/ml) where results are shown in Table 3. and 5.43 (1H, t, J =9Hz,H-3α) assignable to H-3 protons in both α and In this study, the isolation and identification of the major β anomers is reflecting galloylation at C-2 and C-3 of the glucose moiety. phytoconstituents of the leaves of E × neillii of polyphenolic nature Spectrum of 13C-NMR showed the presence of a set of double signals for was carried out for the first time. Chromatographic separation of the the glucose and galloyl carbons. Comparison with previously reported defatted methanolic extract showing the highest phenolic content, data for galloyl glucose assigned an α and β effect resulting from ester- yielded seven phenolic compounds identified for the first time in ification of the sugar OH group. All other resonances were in agreement E × neillii and Erythrina genus except for vitexin (7) which was previ- with the proposed structure (Nawwar et al., 1984). ously isolated from Erythrina caffra (El-Masry et al., 2010). Those com- Kaempferol-3-O-(6″-p-coumaroyl-β-glucopyranoside)(4) pounds included, two hydrolysable tannins (1,3), one phenolic acid showed a pseudomolecular ion peak [M-H]− at m/z 593 corresponding

(2) and four known flavonoids (4–7) represented in Fig. (1). to a molecular formula of C30H26O13. Fragment at m/z 285 denotes a kaempferol aglycone, while a fragment at m/z 323 denotes a coumaroyl 3.1. Identification of compounds glucoside, further confirmed by a fragment at m/z 119. The 1H-NMR spectrum showed the presence of the aromatic protons which appeared

2,3-O-hexahydroxydiphenoyl-(α/β)-glucopyranose (1) showed a as A2 B2 doublets at δH ppm 8.07 (2H, d, J =8Hz,H-2′-6′), 6.96 (2H, dd, precursor ion peak [M–H]− at m/z 481 and fragment at m/z 301 [M– J =2,8Hz,H-3′-5′), beside two aromatic protons meta coupled doublets − H-180] assigned to be [ellagic-H] characteristic for ellagitannins of at δH ppm 6.35 (1H, d, J = 2 Hz, H-8) and 6.30 (1H, d, J = 2 Hz, H-6), and hexahydroxydiphenoyl (HHDP) group. The ellagic acid moiety was con- the absence of signal at δH ppm 6.1 confirming a kaempferol with the at- 1 firmed by fragments at m/z 300, 275 and 249. The H-NMR spectrum of tachment of sugar at C-3. Additional signals at δH ppm 7.47 (2H, d, J = compound (1) showed the presence of two distinctive singlets in the ar- 8Hz,H-2″′-6″′)and6.81(2H,d,J = 8 Hz, H-3″′-5″′) are related to a omatic region at δ- ppm 6.35 and 6.45 (s, H-3 and H-3′) assignable to coumaroyl attachment. This was confirmed by the corresponding sig- 13 hexahydroxydiphenoyl (HHDP) moiety while the presence of two dou- nals in the C-NMR spectrum with peaks at δC ppm 115–145–130-160 blets in the aliphatic region at δH 5.25 (1H, d, J = 3.5 Hz, H-1) and 5.11 and 166 (Alves et al., 2012). Location of the p-coumaroyl moiety at C-6 (1H, d, J = 8 Hz, H-1′), is attributable to the free α-andβ-anomeric of the glucose clearly followed from a significant downfield shift of the

signal of that carbon (δC 62 ppm) as well as on the basis of the downfield shift of the methylenic glucose protons H-6″ in comparison with those of Table 3 the related signals in the reported spectrum of kaempferol-3-O-β-gluco- Radical scavenging activity (IC ) and cytotoxicity (IC ) for the total methanolic extract 50 50 side. The attachment of β glucose at position 3 of the flavonol and its fractions of E × neillii leaves. (kaempferol) was confirmed by the upfield shift of C3 to δC ppm 134.6. Test sample Radical scavenging activity Cytotoxicity Comparison with the reported literature affirmed the structure as μ μ (IC50 g/ml) (IC50 g/ml) Kaempferol-3-O-(6″-p-coumaroyl-β- glucopyranoside)(Slimestad Total methanolic extract 25.2 ± 0.58 55.24 ± 2.67 et al., 1995). N Petroleum ether extract 31.25 ± 0.62 75.34 ± 4.56 The ESI mass spectrum of compounds (5) showed [M-H]− at m/z Methylene chloride extract 31.25 ± 1.84 60.07 ± 1.83 Ethyl acetate extract 17.2 ± 0.73 50.09 ± 2.63 583 corresponding to a molecular formulae C28H24O14 and character- Butanol extract 19.3 ± 0.88 40.92 ± 3.54 ized as a flavone derivative on the basis of its UV absorption (λmax Trolox 15.6 ± 1.63 n.d 366 nm), beside the appearance of the isolated singlet at δH ppm 6.09 Etoposide n.d 1.52 (3-H). Analysis of the 1H-NMR spectrum showed aromatic B-ring pro-

Results are given in mean ± SD of three independent experiments. ton appearing as two aromatic sets (A2B2)atδH ppm 8.11 and 6.93 474 S.K. Gabr et al. / South African Journal of Botany 121 (2019) 470–477

OH OH 46 46 OH HO 5 5 O O HO 2 O 3 2 1 3 9 O 7'' OH O OH HO OH 1 8 O 1 10 O HOOC 1'' O 7' CO OC 7 2 4b 2'' 2' 2'' 3'' 10a 6'' 1' 3' 6a 3'' 5'' 3 2' 6' 4' 1' 1'' 6 HO OH HO OH 4'' 4 4a 4'' 3' 5' 6' OO 5'' 5 5' 6'' OH HO 4' OH O HOOH HO OH OH

2,3-O-hexahydroxydiphenoyl Brevifolin carboxylic acid (2) Nilocitin (3) -(α/β)- glucopyranose (1)

3' OH HO HO 2' 4' 4'' 6'' 8 1 HO 4'' 6'' HO HO O 1' 5' OH OH 3'' 3'' O 5'' 7 2 6' O 5'' O 6''' O OH 6 3 6''' HO 7''' HO 5''' 7''' 2'' 5''' 2'' 3' O 3' 5 4 O 1''' O OH 1''' OH 2' 1'' 2' 4'' 1'' 4' 4''' 4' 1'' 2'' 2''' 8 OH O OH 8 1' HO HO O 1' HO HO O O 3'' 5' 3''' 5' 2'' 7 2 7 2 6' OH 6' 3''' 3'' OH 6 3 HO 5'' 6 4 3 4 6'' OH 5 5 4''' 2''' 4'' 1''' OH O OH O 5''' O OH 6'''

Kaempferol-3-O-(6"-p- 2"-O-gallpyl vitexin (5) 2"-O-galloyl orientin (6) coumaroyl-β- glucopyranose (4)

HO 4'' 6'' OH

3'' 5'' HO 2'' O 3' OH 2' H 1'' 4' 1 HO 8 1' 7 O 5' 2 6'

6 3 5 4

OH O Vitexin (7)

Fig. 1. Chemical structures of compounds 1–7. denoting 2′,6′ and 3′,5′ respectively, a sharp downﬁeld singlet inte- proton signals are attributable to a β-D-glucopyranosyl moiety. The grated for two protons appeared at δH ppm 6.74 which denotes the deshielding of 2″ H of the glucosyl residue which appeared at δH ppm magnetically equivalent 2 and 6 protons of a galloyl group. The aliphatic 5.50 (t) relative to the non galloylated analog vitexin indicates that the OH at this position was acylated. 13C-NMR conﬁrmed the glucosyl

Table 4

Radical scavenging activity (IC50) for the isolated compounds of E × neillii leaf extract. Table 5 Docking score (Kcal/mol) of the isolated phenolics from the aqueous methanolic extract of μ Test sample IC50 g/ml E ×neilliiwithin the active site of Heme oxygenase (1N3U) calculated by MOE. 2,3 O-hexahydroxydiphenoyl-(α/β)-glucopyranose 9.73 ± 0.58 Ligand Energy scores (Kcal/mol) Brevifolin carboxylic acid 5.12 ± 0.84 Nilocitin 4.56 ± 1.84 2, 3-O-hexahydroxydiphenoyl-(α/β)-glucopyranose −11.9851 Kaempferol 3-O-(6″-p-coumaroyl-β- glucopyranoside 6.6 ± 0.95 Brevifolin carboxylic acid −10.7644 2″-O-galloyl vitexin 3.72 ± 0.76 Nilocitin −16.3531 2″-O-galloyl orientin 1.85 ± 0.28 Kaempferol-3-O-(6″-p-coumaroyl-β- glucopyranoside) −13.0487 Vitexin 10.5 ± 0.42 2″-O-Galloyl vitexin −18.1335 Trolox 15.6 ± 1.63 2″-O-Galloyl orientin −18.6837 Vitexin −10.8081 Results are given in mean ± SD of three independent experiments. S.K. Gabr et al. / South African Journal of Botany 121 (2019) 470–477 475

Brevifolin carboxylic acid 2, 3-O-hexaydroxydiphenoyl-(a/b)- glucopyranose

¢¢ b Nilocitin Kaempferol-O-(6 -p-coumaroyl- -glucopyranoside

2¢¢-O-galloyl vitexin 2¢¢-O-galloyl orientin

Side chain acceptor/donor

Backbone acceptor/donor

Blue shadow represents ligand exposure

Vitexin

Fig. 2. Binding residues of the isolated compounds with heme oxygenase (PDB: (1N3U)).

attachment at C-8 by a significant downfield shift appearing at δC ppm et al., 2010) with absence of H-8. The downfield shift of C-8 to δC ppm 104.60 compared with the non-glycosylated derivative. 104.67 and C1″ of glucose at δC ppm 73.48 confirmed the attachment 13 The C-NMR downfield shift of the glucose carbon C 2″ (δC 73 ppm) of glucose moiety at C-8, therefore identified as vitexin (Latté et al., 1 13 in comparison with that of the corresponding carbon in free vitexin (δC 2002). Hand C- NMR data of compounds (4–7) are displayed in 70.85 ppm) (Agrawal et al., 1989) accompanied by the upfield shift of Tables 1 and 2. the anomeric carbon resonance (at δC 72 ppm) indicated that this position was substituted. Therefore compound (5) was identified as 2″-O- 3.2. Oxygen radical scavenging capacity (ORAC) assay for isolated Galloyl vitexin. compounds Compound (6)showed[M-H]− at m/z 599 corresponding to a molecular formula C28H24O15. Fragment at m/z 429 denoted the loss of a Due to the detrimental effects of oxidative stress in the different ail- gallic acid moiety (−170), while a fragment at m/z 309 [M-H-170- ments and their role in the pathogenesis of cardiovascular disease, this 120] denotes the loss of a glucose moiety (Latté et al., 2002). 1H-NMR study was designed to verify the antioxidant activity of the isolated spectrum revealed similar features to compound (5) except for the compounds using ORAC assay (Table 4) besides justifying this activity

ABX-spin systems for the B-rings instead of the aromatic A2B2 previ- with the molecular modeling to identify the binding interactions as ously observed denoted by a doublet specificfor2′ at δH ppm 7.45 and shown in Table 5. Investigating the radical scavenging activity using 1 13 a doublet of doublet denoting H-6′ at δH ppm 7.56. Hand C-NMR ORAC assay resulted in the prevalence of 2″-O-galloyl orientin, which spectra displayed the typical signals for the spectra of flavones with showed the highest antioxidant activity with IC50 1.85 μg/ml followed C-8-hexosyl substituent (Latté et al., 2002; Rabe et al., 1994).Thus com- by 2″-O-galloyl vitexin with IC50 3.72 μg/ml while the lowest activity pound (6)wasidentified as 2″-O-Galloyl orientin. was observed with vitexin (IC50 10.5 μg/ml). Compound(7) appeared as the non-galloylated derivative of com- Several reports showed that the radical scavenging capacities in- pound (5), showing typical signals of apigenin derivative (El-Masry creased with an increase in the number of phenolic hydroxyl groups; 476 S.K. Gabr et al. / South African Journal of Botany 121 (2019) 470–477 this was observed for the three classes of compounds: flavonoids, pattern for the most active compounds. It was reported that Met 34 (a galloyl glucoses and ellagitannins (Fortes et al., 2015). In our study, polar thiol group) in the hydrophobic pocket of the heme structure the attachment of gallic acid to orientin, vitexin and its presence in had a strong binding with electronegative moieties giving rise to a po- nilocitin justified a higher antioxidant capacity compared with the tent activity, in addition to a role in the stabilizing/destabilizing interac- non-galloylated compounds. Orientin higher scavenging activity com- tions (Rahman et al., 2012). This virtual screening study justified the pared to vitexin is attributed to the two hydroxyl groups in ring B higher antioxidant activity of the tested compounds and their ability (Dimitrić Marković et al., 2017), while the high activity of brevifolin car- to provide a direct and indirect antioxidant effects through their radical boxylic acid and 2,3-HHDP-glucopyranose may be attributed to the car- scavenging activity and an induction of HO-1. boxylic group beside the phenolic hydroxyl groups (Chang et al., 2017; Karamac et al., 2005). 4. Conclusion The role of phenolic compounds as radical scavengers has been well studied. However, another mechanism for antioxidants that has been This study highlighted the importance of E × neillii that can repre- the target for most of recent researches is compounds that can indirectly sent a promising effective candidate to combat oxidative stress due to upregulate endogenous antioxidant defenses, allowing profound anti- its phenolic constituents. Additionally, our findings highlight that dur- oxidant protection. Those compounds have long half-lives, and are un- ing the design of a novel antioxidant the conserved amino acids have likely to evoke pro-oxidant effects, therefore providing a more to be considered for enhancing the activity of the phytoconstituents efficient and long lasting response to oxidative stress. (Barbagallo against HO-1. et al., 2012). The results of the in-vitro antioxidant potential of the isolated compounds using ORAC assay showed pronounced activity as rad- Conflict of interest ical scavengers. Based on this, a molecular docking study was performed to document the mechanism of action of the isolated flavonoids and The authors declare that there are no conflicts of interest. ellagitannins as inducible for HO-1. Funding sources 3.3. Molecular docking This research did not receive any specific grant from funding agen- HO-1 is an enzyme playing a vital role in the oxidative stress (heme cies in the public, commercial, or not-for-profit sectors. oxygenase). Upregulation of heme oxygenase-1 (HO-1), a phase II de- toxifying enzyme in endothelial cells, is considered to be helpful in car- Acknowledgements diovascular disease. In addition, HO-1 could exert cytoprotective effect by preventing apoptosis (Lian et al., 2008). Understanding the heme The authors are thankful for the Pharmaceutical Chemistry Depart- proteins structures and heme binding environment provide valuable ment, Faculty of Pharmacy, MSA University for assisting in the molecu- fl guidelines in the design of novel antioxidants and anti-in ammatory lar docking study. (Choi and Alam, 1996). Molecular docking study of all compounds was carried out on HO-1 (PDB code: 1N3U) to correlate the isolated Appendix A. Supplementary data compounds with the demonstrated activity through determining the interactions of these compounds within the active site of HO-1. Supplementary data to this article can be found online at https://doi. fl The co-crystallized ligand (Heme) was exibly re-docked to verify the org/10.1016/j.sajb.2018.12.011. docking protocol using MMFF94 force field. The intermolecular interactions between the ligand and the target receptor was evaluated. 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