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 × neilliipolyphenolics

S.K. Gabr a, R.O. Bakr a,⁎, E.S. Mostafa a, A.M. El-Fishawy b, T.S. El-Alfy b

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

⁎ Corresponding author.E-mail addresses: sgabr@msa.eun.eg (S.K. Gabr), roma

emostafa@msa.eun.eg (E.S. Mostafa), elfishawyahlam49@ahlam.elfishawy@pharma.cu.edu.eg (A.M. El-Fishawy), ta(T.S. El-Alfy).

https://doi.org/10.1016/j.sajb.2018.12.0110254-6299/© 2018 SAAB. Published by Elsevier B.V. All ri

a b s t r a c t

a r t i c l e i n f o

Article history:Received 24 August 2018Received in revised form 9 December 2018Accepted 18 December 2018Available online xxxx

Edited by J Grúz

Species of genus Erythrina have a great contribution in folkmedicine; various species are utilized as a tranquilizer,to treat insomnia, inflammation and colic. Besides, Erythrina species have reported antioxidant, hepatoprotectiveand anxiolytic activities. Erythrina × neillii is a hybrid obtained through a cross between E. herbacea L. andE. humeana Spreng. It has not been well-studied for its chemical or biological profile; therefore it represents aninteresting field of study. In this study, seven phenolic compounds; two hydrolysable tannins (1,3), one phenolicacid (2) and four known flavonoids (4–7) were isolated and characterized for the first time in E × neillii andErythrina genus except for vitexin (7). Isolated compounds were assessed for their antioxidant activities usingORAC assay. 2″-O-galloyl orientin (6) exhibited the highest activity followed by 2″-O-galloyl vitexin (5). Flexiblemolecular docking on heme oxygenase, an important stress protein that is involved in cellular protection, antiox-idant and anti-inflammatory activities, justified the antioxidant activity of the isolated compounds. The best scor-ing was observed with 2″-O-galloyl orientin forming four binding interactions with residues, Arg 136 (twointeractions), Met34 and Gly139. Erythrina × neillii offered powerful and available antioxidant beside signifi-cantly active phytoconstituents.

© 2018 SAAB. Published by Elsevier B.V. All rights reserved.

Keywords:Erythrina × neilliiPolyphenolicsORACDockingHeme oxygenase

1. Introduction

Oxidative stress (OS) is an imbalance between the reactive oxygenspecies (ROS) formation and the antioxidant defense mechanisms. Attheir high concentrations, ROS can react with differentmacromolecules,therefore involved many disease processes. In our body, the cellularantioxidant defense systems including glutathione (GSH), and ROS-scavenging enzymes, such as superoxide dismutase (SOD), catalaseand glutathione peroxidase (GPX) regulate the levels of ROS (Valkoet al., 2007).

Heme oxygenase-1 (HO-1) is a cellular stress protein that plays animportant role in the oxidative catabolism of heme leading to theformation of biliverdin (BV), free iron and carbon monoxide (CO).Whereby, BV formed is rapidly converted to the strong antioxidantbilirubin (BR), which is then converted back into BV through reactingwith ROS allowing their neutralization. Therefore, HO-1 has its potentialability to regulate oxidative and inflammatory which contribute to anefficacy in controllingmetabolic diseases and make it a target of several

r@msa.eun.eg (R.O. Bakr),gmaireferencesl.com,ha.elalfy@pharma.cu.edu.eg

ghts reserved.

researches (Kapitulnik and Gonzalez, 2004; Pae et al., 2010; Son et al.,2013). Plant phenolics have beneficial effects against many pathologicalconditions including oxidative stress as they are able to scavenge freeradicals (Brewer, 2011).

Erythrina is a genus of flowering plants in the pea family (Fabaceae).It contains more than 200 species distributed worldwide and known as“coral tree” (Neill, 1988). Traditionally, plants of this genus have a vari-ety of uses such as tranquilizer, anti-inflammatory, in treatment of colicand liver ailments (Chhabra et al., 1984; García-Mateos et al., 2001;Ghosal et al., 1972). Erythrina is very rich in its phytoconstituents includ-ing alkaloids, flavonoids with its different classes, cinnamoylphenols,stilbenoids, 3-phenoxychromones, coumastans, 3-phenyl-coumarins,lignans, cinnamate esters, triterpenes, sesquiterpenes, long-chain car-boxylic acids and long-chain alcohols (Callejon et al., 2014; Chachaet al., 2005; Majinda et al., 2005; Nkengfack et al., 2001; Pérez et al.,2015; Wanjala and Majinda, 2000; Yenesew et al., 2003). A wide rangeof biological activities has been investigated including, antimicrobial ac-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;Nkengfack et al., 2001; Setti-Perdigão et al., 2013).

Erythrina × neillii Mabberley & Lorence is a hybrid derived from thecross between E. herbacea and E. humeana. In continuation of our earlierstudies on the pharmacognostical and genetic properties of this plant

471S.K. Gabr et al. / South African Journal of Botany 121 (2019) 470–477

(Gabr et al., 2017), this study was undertaken to characterize the mainactive constituents in E × neillii leaf extract based on their spectroscopicdata and comparison with reported literature. Those compounds weretested for the in-vitro antioxidant activity using oxygen radical absor-bance capacity (ORAC) assay, moreover, flexible docking on HO-1 justi-fied this activity. To the best of our knowledge, this is the first report fora chemical characterization of E × neillii and its activity.

2. Material and methods

2.1. General

Column chromatography (CC) was carried on, polyamide S-6(E-Merk), Sephadex LH-20 (Pharmacia, Uppsala, Sweden), MCI gelcolumn (CHP-20P, 75–150 μm; Mitsubishi Chemical Co., Dusseldorf,Germany). For paper chromatography, Whatman No. 1 and 3 sheetswere used (E-Merk). Solvent systems (S1) BAW (Bu-HOAC-H2O,4:1:5, upper layer), (S2) 15% HOAC. Detection of the developedchromatogramswas performed under UV 254 and 365 nm light and ex-posure to ammonia vapor. The NMR spectra were acquired in CD3OD ona Jeol ECA 500MHz using tetramethylsilane (TMS) as the internal stan-dard. UV spectrophotometer (Shimadzu UV/VIS-1601) was used foranalysis of pure samples in MeOH and in different UV shift reagents.ESI-MS wasmeasured on spectrometer MAT 95 (Finigan-MAT, Bremen,Germany). Oxygen radical absorbance capacity (ORAC) was performedon fluorometer, FLUO star OPTIMA, Franka Ganske, BMG LABTECHassisted by a Shimadzu UV–Visible-1601 spectrophotometer. For cyto-toxic assay: Optical density was measured on a plate reader (FluostarOmega, BMG Labtech, Offenburg, Germany).

2.2. Reagents, chemicals and cells

Fluorescein (Sigma–Aldrich), Etoposide (Alexis Biochemicals),6-hydroxy-2, 5, 7, 8-tetra-methylchroman-2-carboxylic acid Trolox(Sigma–Aldrich), 2,2′-azobis (2-amidinopropane) dihydrochloride(AAPH) (Sigma–Aldrich). All solvents used in column chromatographywere analytical grade. Human bladder carcinoma cell line 5637was ob-tained from CLS Cell Lines Service (Eppelheim, Germany). Cells werecultured in RPMI 1640 medium (BioWhittaker, Lonza, Verviers,Belgium) supplemented with 10% fetal bovine serum (Sigma–Aldrich,Taufkirchen, Germany) and antibiotics (100 U/ml penicillin, 100 μg/mlstreptomycin; Sigma Aldrich, Taufkirchen, Germany) at 95% humidity,5% CO2 and 37 °C.

2.3. Plant material

Leaves of Erythrina × neilliiMabberley & Lorencewere collected dur-ing the flowering stage, October 2012 from El-Zohria garden, Cairo andwere kindly identified byDr. Gwilym P. Lewis, Legume Research Leader,Comparative Plant & Fungal Biology, Royal Botanic Gardens, Kew, UK. Avoucher specimen was deposited in the Herbarium of the Faculty ofPharmacy, MSA University under registration number (RS020) and atthe Department of Pharmacognosy, Faculty of Pharmacy, Cairo Univer-sity (no.1-11-2012).

2.4. Extraction and fractionation of extracts for quantitative estimation,ORAC and cytotoxic assay

Two hundred grams of powdered leaves were extracted with 70%methanol then fractionated beginning with Pet. ether (40–60 °C)followed by methylene chloride, ethyl acetate and butanol. The extrac-tion with each solvent continued till exhaustion. Each extract wasdistilled off under reduced pressure and dried to constant weight thenkept for determination of the total phenolic and flavonoid contents aswell as the antioxidant and cytotoxic activities.

2.5. Quantitative estimation of phenolic and flavonoid contents

The total phenolic content of each fraction was determined by theFolin–Ciocalteau Reagent (FCR) using gallic acid as standard andexpressed as milligram of gallic acid equivalents (GAE) per gram dryextract (Sellappan et al., 2002). The absorbance was measured at λmax

765 nm using shimadzu UV–visible spectrophotometer (1800 UV-probe) after incubation for 2 h at room temperature. The total flavonoidcontent was determined by aluminum chloride colorimetric assaybased on the quercetin calibration curve and expressed as milligramof quercetin equivalent per milligram dry extract (QE). The absorbancewas measured at λmax 415 nm (Kosalec et al., 2004). Measurementswere carried out in triplicate.

2.6. Determination of oxygen radical absorbance capacity (ORAC) assay

The evaluation of the oxygen radical absorbance activity by ORACwas performed for the total extract and its fractions as well as the iso-lated compounds using a 96-well microplate reader as previouslyreported (Huang et al., 2002; Nawwar et al., 2012). The antioxidant ca-pacity of the isolated compounds was measured by determining thetime course of the fluorescence decay of fluorescein, induced by 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) compared with avitamin E derivative, 6-hydroxy-2, 5, 7, 8-tetra-methylchroman-2-carboxylic acid (Trolox), used as a positive control.

2.7. Neutral red uptake (NRU) cytotoxicity assay

Human bladder carcinoma cells were sub-cultured twice aweek andregularly tested for mycoplasma contamination. Cytotoxicity of thetested extracts was investigated using the neutral red uptake (NRU)assay as described before (Repetto et al., 2008). The optical densitywas measured at 450 nm in a plate reader while all the experimentswere tested in duplicates. The IC50 values were obtained from thedose–response curves and expressed in mean ± SD. Etoposide wasused as positive control.

2.8. Extraction and isolation

The dried powdered leaves (1 kg) were defatted with petroleumether then subjected to extensive extraction using 70% methanolunder reflux (5 L × 5, 60 °C) till exhaustion. The defatted methanolicextract was concentrated under reduced pressure to yield a viscous res-idue (200 g). The residuewas precipitated fromH2O using excessmeth-anol (1:10) followed by evaporation of the filtrate under vacuum toafford 150 g. The dried extract was subjected to CC using polyamide(500 g, 100 × 5 cm) beginning with H2O then decreasing the polaritygradually using MeOH from 100% H2O to 100% MeOH) to afford 50fractions (500ml each), which were collected into 6 collective fractions(I–VI) based on paper chromatographic investigation assisted by UV-light and spray reagents (Marzouk et al., 2009). Fraction I was devoidof polyphenolic compounds. Compound (1) (12 mg) was obtainedfrom fraction II (2.05 g) eluted with 10% methanol and further purifiedon sephadex LH-20 and water saturated n-BuOH as eluent. Compound(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 elutedwith water saturated n-BuOH to afford two individual sub-fractions,whereas compound (2) was isolated from sub-fraction (ii). Compound(3), (22 mg) was isolated from fraction IV (5.41 g), eluted with 30%MeOH and purified over Sephadex LH-20 using 50% MeOH. Fraction V(2.78 g) eluted with 40% MeOH was isolated using preparative PC andS1 then finally purified on a sephadex LH-20 eluted with 50% MeOHto yield compound (4) (15mg). The repeated preparative PC of fractionVI (3.2 g, eluted with 70% MeOH), and S1 as an eluent afforded com-pounds 5 (20 mg), 6 (15 mg) and 7 (10 mg), each was individually pu-rified on Sephadex LH-20 column using 50% aqueous MeOH.

Table 11H-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.

4 5 6 7

AglyconeH-3 6.09 6.52 6.57H-6 6.3 (d; 2) 6.08 6.09 6.51H-8 6.35 (d; 2) – – –H-2′ 8.07 (d; 8) 8.11 (d;8) 7.45 (d; 2) 8.12 (d; 7.5)H-3′ 6.96 (dd; 2,8) 6.93 (d; 8) – 6.82 (d; 7.5)H-5′ 6.96 (dd; 2,8) 6.93 (d; 8) 6.89 (d; 8) 6.82 (d; 7.5)H-6′ 8.07 (d; 8) 8.07 (d; 8) 8.11 (d; 8) 7.56 (dd; 8, 2) 8.12 (d; 7.5)

GlucosylH-1″ 4.92 (d, 7) 4.97 (d;8) 4.97 (d; 8) 4.99 (d; 9.6)H-2″ 5.50 (t;9.3) 5.52 (t; 9.5)H-3″ 3.71 (t;9.3) 3.71 (t; 9.1)H-4″H-5″H-6″ 4.61 (Br.d; 10)

GalloylH-2″′, 6″′ 6.74 (s) 6.74 (s)Coumaroyl2″′, 6″′ 7.47 (d; 8)3″′, 5″′ 6.81 (d; 8)7″′ 7.81 (d; 15)8″′ 6.45 (d; 15)

Table 213C-NMR spectral data (125 MHz; CD3OD) of compounds 4–7.

4 5 6 7

C-2 157.04 166.60 166.60 164.00C-3 134.60 102.70 102.70 102.49C-4 178.19 182.99 182.10 183.00C-5 160.40 161.70 161.70 161.00C-6 98.92 98.99 98.10 97.58C-7 165.00 163.30 163.30 157.60C-8 92.81 104.60 104.61 104.67C-9 157.04 157.00 157.03 156.05C-10 105.16 102.70 102.70 102.98C-1′ 121.34 122.00 122.00 122.00C-2′ 130.91 129.10 113.70 128.68C-3′ 115.88 115.90 145.90 115.98C-4′ 160 161.70 150.00 161.18C-5′ 115.88 115.90 115.70 115.98C-6′ 130.91 129.10 120.00 128.68

GlucosylC-1″ 97.84 72.00 72.00 73.48C-2″ 72.02 73.00 73.01 70.93C-3″ 77.25 77.00 76.89 77.73C-4″ 70.80 71.50 7.40 71.50C-5″ 75.50 82.20 82.20 81.43C-6″ 62.50 61.80 61.80 61.38

GalloylC-1″′ 120.00 120.00C-2″′, C-6″′ 109.20 109.20C-3″′, C-5″′ 145.20 145.20C-4″′ 136.68 136.98C-7″′ 167.23 167.70

CoumaroylC-1″′ 125.94C-2″′, C-6″′ 130.91C-3″′, C-5″′ 115.57C-4″′ 160.00C-7″′ 145.34C-8″′ 115.81C=O 166.32

472 S.K. Gabr et al. / South African Journal of Botany 121 (2019) 470–477

2.9. Isolated compounds

2,3-O-hexahydroxydiphenoyl-(α/β)-glucopyranose (1):off-whiteamorphous powder; UV: λmax(MeOH): 259. M, 482, ESI-MS (negativemode) m/z 481 [M-H]−, 301 [M-H-180] -, 300 [ellagic acid-H]−, 275and 249.1H-NMR: Glucose moiety in α-anomers: δ ppm 5.25 (1H, d,J = 3.5 Hz, α-H-1), 4.49 (1H, dd, J = 3.5 Hz, α-H-2), 3.71–4.56 (m, H-4-H-6α). Glucose moiety in β-anomers: 5.11 (1H, d, J = 8 Hz, β- H-1),4.69 (1H, dd, J = 8 and 7.5, β-H-2), 4.84 (1H, t, J = 7.5 Hz, β-H-3),3.71–4.56 (m, H-4-H-6β). Hexahydroxy-diphenoyl moiety in α- & β-anomers: 6.35 (2H in total, s, H-3'α/β HHDP), 6.45 (1H, s, H-3″α/βHHDP).13C-NMRglucose moiety: 61.04 (C-6 α/β), 67.40 (C-4 α),67.53 (C-4 β), 72.08 (C-2 α), 74.41 (C-2 β), 77.42 (C-3 α), 77.43 (C-5α/β), 80.08 (C-3 β), 90.17 (C-1 α), 93.48 (C-1 β). Hexahydroxy-diphenoyl moiety in α-&β-anomers: 105.20, 105.53 (C-3′ & C-3″),113.92, 114.16 (C-1′ & C-1″), 125.25, 125.56 (C-2′ & C-2″), 135.25,135.38 (C-5′ & C-5″),144.63, 144.70, 145.10, 145.29 (C-4′, C-4″ and C-6′ & C-6″), 169.09, 169.52 (C=O).

Brevifolin carboxylic acid (2):Off-white amorphous powder; UV:λmax (MeOH) 278, 350, 362; M, 292, ESI-MS (negative mode) m/z 291[M-H]−, 247 [M–H-CO2]. 1H-NMR: δ ppm 2.51 (2H, s, H-3 and H-3′);3.75 (1H, dd, J = 4 and 2 Hz, H-2); 6.95 (1H, s, H-7). 13C-NMR: δ ppm39.36 (C-3); 40.32 (C-2); 109.19 (C-7); 121.21 (C-4b); 121.21 (C-6a);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).

2,3-digalloyl-(α/β)-4C1-glucopyranose (nilocitin) (3):Off-whiteamorphous powder; UV: λmax (MeOH) 274; M, 484, ESI-MS (negativemode) m/z 483 [M-H]−, 331 [M–H-galloyl]−, 169 and 125.1H-NMR:glucose moiety: δ ppm 3.6–3.95 (m, H-4α and H-4β, 6-Hα and 6-Hβ),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(1H, dd, J = 9.5 and 9 Hz, H-3β). Galloyl moieties in α- andβ-anomers: δ ppm 6.87(2H, s, H-2′, 6′), 6.88 (2H, s, H-2″,6″).13C-NMR: α-glucose moiety: δ ppm 60.6 (C-6), 65.13 (C-4), 73.20(C-2, 3 and 5), 89.79, 94.85 (C-1 α/β). β-glucose moiety: δ ppm 60.58(C-6), 68.79 (C-5 α/β), 68.82 (C-4),73.83 (C-2), 75.50 (C-3), 94.50(C-1). Galloyl moieties in α- and β-anomers: δ ppm 109.29(C-2′,6'α/β, 2″,6″ α/β), 119.02120.64, 121.35 (C-1′ α/β,1″ α/β), 138.67,139 (C-4'α/β,4″α/β), 145.66 (C-3′,5′ α/β,3″,5″ α/β), 165, 165.2, 165.4,166 (C=O α/β, C′ = O α/β).

Kaempferol-3-O-(6″-p-coumaroyl-β-glucopyranoside) (4):Brown amorphous powder; UV: λmax(MeOH): 266, 369; (NaOMe)273, 325, 398; (NaOAc) 271, 302 (shoulder), 356; (NaOAc/H3BO3)270, 365; (AlCl3) 270, 345, 405; (AlCl3/HCl) 270, 342, 403.M, 594, ESI-MS (negative mode) m/z 593 [M-H]−, 323, 285, and 119.1H-NMR and13C-NMR (Tables 1 and 2).

2″ O-galloyl vitexin (5): yellow amorphous powder. UV: λmax

(MeOH): 273, 333; (NaOMe) 283, 302 (shoulder), 386; (NaOAc) 283,392; (NaOAc/H3BO3) 275, 334; (AlCl3) 280, 306, 349, 386; (AlCl3/HCl)280, 305, 344. ESI-MS (negative mode) m/z 583 [M-H]−, 431 [M-H-galloyl]−, 169. 1H-NMR and 13C-NMR (Tables 1 and 2).

2″ O-galloyl orientin (6): Yellow amorphous powder, ESI-MS (neg-ative mode) m/z 599 [M-H]−, 429 [M-H-gallic acid]−, 447 [M-H-galloyl]−, 169. 1H-NMR and 13C-NMR (Tables 1 and 2).

Vitexin (7): UV: λmax(MeOH): 273, 333; (NaOMe) 275, 300 (shoul-der), 340; (NaOAc) 276, 352; (NaOAc/H3BO3) 275, 334; (AlCl3) 280,306, 349, 386; (AlCl3/HCl) 280, 306, 349, 386.M, 432, ESI-MS (negativemode)m/z 431 [M-H]−1H-NMR and 13C-NMR (Tables 1 and 2).

2.10. Molecular docking

Flexible molecular docking study was carried out on Heme oxygen-ase for the isolated compounds from the aqueous methanolic extract ofE × neillii using Molecular Operating Environment (MOE) software(MOE_2014.0901). The X-ray crystal structure of Heme oxygenase

(PDB code: 1N3U) was downloaded from the Protein Data Bank(www.pdb.org). The predicted binding energies for the different com-pounds as well as the binding interactions were determined andcompared.

473S.K. Gabr et al. / South African Journal of Botany 121 (2019) 470–477

3. Results and discussion

Erythrina × neillii methanolic extract was fractionated with petro-leum ether (PE), methylene chloride (MC), ethyl acetate (EA) and buta-nol (Bu) till exhaustion. The resultant pooled extracts were separatelyconcentrated under reduced pressure and kept for determination ofthe phenolic and flavonoid contents beside the antioxidant and cyto-toxic activities. The phenolic content was estimated as gallic acid equiv-alent (GAE) (y=0.0111×−0.0339, R2=0.9901). The totalmethanolicextract showed the highest content (10.12 mg/g) followed by the EA(9.56 mg/g) then Bu fraction (9.34 mg/g), while the lowest concentra-tion was observed with MC extract (9.1 mg/g). Whereas, the flavonoidcontent was estimated based on the complex formed with aluminumchloride and quantified as quercetin equivalent (QE) (y = 0.0047×,R2 = 0.9637). Bu showed the highest flavonoid content (15.9 mg/gQE) followed by the EA (13.5mg/gQE) then the totalmethanolic extract(12.07 mg/g QE), while theMC fraction (7.34mg/g) showed the lowestconcentration.

ORAC assay helped in determination of the antioxidant activitywhere, the fluorescence decay was detected and the % inhibition ofthe decay was calculated (Table 3). The antioxidant activity of all ex-tracts increased in a dose-dependent manner where, EA fractionshowed the highest activity followed by Bu, which may be attributedto the high concentration of phenolic contents. While the PE and MCfractions showed the lowest activity.

The potential cytotoxicity of the E × neillii total methanolic extractand its fractions was evaluated on human bladder carcinoma cell line5637 using Neutral Red Uptake (NRU) assay (Repetto et al., 2008). Bushowed the highest cytotoxicity (IC50 of 40.92 μg/ml) followed by EA(IC50 of 50.09 μg/ml) where results are shown in Table 3.

In this study, the isolation and identification of the majorphytoconstituents of the leaves of E × neillii of polyphenolic naturewas carried out for the first time. Chromatographic separation of thedefatted methanolic extract showing the highest phenolic content,yielded seven phenolic compounds identified for the first time inE × neillii and Erythrina genus except for vitexin (7) which was previ-ously isolated from Erythrina caffra (El-Masry et al., 2010). Those com-pounds included, two hydrolysable tannins (1,3), one phenolic acid(2) and four known flavonoids (4–7) represented in Fig. (1).

3.1. Identification of compounds

2,3-O-hexahydroxydiphenoyl-(α/β)-glucopyranose (1) showed aprecursor ion peak [M–H]− at m/z 481 and fragment at m/z 301 [M–H-180] assigned to be [ellagic-H]− characteristic for ellagitannins ofhexahydroxydiphenoyl (HHDP) group. The ellagic acidmoietywas con-firmed by fragments atm/z 300, 275 and 249. The 1H-NMR spectrum ofcompound (1) showed the presence of two distinctive singlets in the ar-omatic region at δ- ppm 6.35 and 6.45 (s, H-3 and H-3′) assignable tohexahydroxydiphenoyl (HHDP) moiety while the presence of two dou-blets in the aliphatic region at δH 5.25 (1H, d, J = 3.5 Hz, H-1) and 5.11(1H, d, J = 8 Hz, H-1′), is attributable to the free α- and β-anomeric

Table 3Radical scavenging activity (IC50) and cytotoxicity (IC50) for the total methanolic extractand its fractions of E × neillii leaves.

Test sample Radical scavenging activity(IC50 μg/ml)

Cytotoxicity(IC50 μg/ml)

Total methanolic extract 25.2 ± 0.58 55.24 ± 2.67Petroleum ether extract N31.25 ± 0.62 75.34 ± 4.56Methylene chloride extract 31.25 ± 1.84 60.07 ± 1.83Ethyl acetate extract 17.2 ± 0.73 50.09 ± 2.63Butanol extract 19.3 ± 0.88 40.92 ± 3.54Trolox 15.6 ± 1.63 n.dEtoposide n.d 1.52

Results are given in mean ± SD of three independent experiments.

glucose protons, respectively (Nawwar et al., 1984). A pair of doubletof doublet at δH ppm 4.49 (1H, dd, J = 3.5, 8 Hz, α-H-2) and at 4.69(1H, dd, J = 3.5, 8 Hz, β-H-2), is assignable to the H-2 glucose proton,in both α- and β- anomers, respectively. It revealed a resonance at δHppm 4.84 (1H, t, J = 7.5 Hz), assignable to the H-3 glucose protons.The downfield H-2 and H-3 sugar resonances beside that of C2 and C3confirmed their attachment to HHDP. Therefore assigning compound(1) to be 2,3-O-hexahydroxydiphenoyl-(α/β)-glucopyranose(Tanaka et al., 1986).

Brevifolin carboxylic acid(2) was obtained as an off-white amor-phous powder with a pseudomolecular ion peak [M-H]− at m/z 291with fragment at m/z 247 [M-H-44]− denoting the loss of carboxylicgroup.1H and 13C-NMR analysis were in agreement with the literaturefor brevifolin carboxylic acid (Nawwar et al., 1994).

2,3 digalloyl-(α/β)-4C1-glucopyranose (nilocitin)(3), appeared asan off-white amorphous powder with a pseudomolecular ion peak[M-H]− at m/z 483. Fragment at m/z 331 denotes the loss of a galloylmoiety [M-H-152]− which was further confirmed by fragments at m/z169 and 125 which are diagnostic of gallic acid moiety. 1H-NMR spec-trum showed the characteristic two proton singlets of the two galloylgroup at δH ppm 6.88 and 6.87 (2H, s, H-2″,6″) in the aromatic regionwhile in the aliphatic region, two different patterns of proton signals be-longing to α/β anomeric mixture of disubstituted glucose appeared. Apair of doublets centered at δH ppm 5.10 (1H, d, J = 3.5 Hz, H-1α) and4.94 (1H, d, J= 9.5, H-1β) assigned the α and β anomeric glucose pro-tons indicating a free anomeric OH group. The downfield glucose pro-tons at δH ppm 4.98 (1H, dd, J = 9.5 and 3.5 Hz, H-2α) and 5.22 (1H,dd, J=9.5 and 3.5, H-2β) assignable to H-2 glucose protons, in additionto the downfield shift at δH ppm 5.522 (1H, dd, J=9.5 and 9 Hz, H-3β)and 5.43 (1H, t, J=9Hz, H-3α) assignable to H-3 protons in bothα andβ anomers is reflecting galloylation at C-2 and C-3 of the glucosemoiety.Spectrum of 13C-NMR showed the presence of a set of double signals forthe glucose and galloyl carbons. Comparison with previously reporteddata for galloyl glucose assigned anα and β effect resulting from ester-ification of the sugar OH group. All other resonances were in agreementwith the proposed structure (Nawwar et al., 1984).

Kaempferol-3-O-(6″-p-coumaroyl-β-glucopyranoside)(4)showed a pseudomolecular ion peak [M-H]− atm/z 593 correspondingto a molecular formula of C30H26O13. Fragment at m/z 285 denotes akaempferol aglycone, while a fragment atm/z 323 denotes a coumaroylglucoside, further confirmed by a fragment at m/z 119. The 1H-NMRspectrum showed the presence of the aromatic protonswhich appearedas A2 B2 doublets at δH ppm 8.07 (2H, d, J=8Hz, H-2′-6′), 6.96 (2H, dd,J=2,8Hz,H-3′-5′), beside twoaromaticprotonsmeta coupleddoubletsat δH ppm6.35 (1H, d, J=2Hz, H-8) and 6.30 (1H, d, J=2Hz, H-6), andthe absence of signal at δH ppm6.1 confirming a kaempferolwith the at-tachment of sugar at C-3. Additional signals at δH ppm 7.47 (2H, d, J =8 Hz, H-2″′-6″′) and 6.81 (2H, d, J = 8 Hz, H-3″′-5″′) are related to acoumaroyl attachment. This was confirmed by the corresponding sig-nals in the 13C-NMR spectrum with peaks at δC ppm 115–145–130-160and 166 (Alves et al., 2012). Location of the p-coumaroyl moiety at C-6of the glucose clearly followed from a significant downfield shift of thesignal of that carbon (δC 62 ppm) aswell as on the basis of the downfieldshift of themethylenic glucoseprotonsH-6″ in comparisonwith those ofthe related signals in the reported spectrumof kaempferol-3-O-β-gluco-side. The attachment of β glucose at position 3 of the flavonol(kaempferol) was confirmed by the upfield shift of C3 to δC ppm 134.6.Comparison with the reported literature affirmed the structure asKaempferol-3-O-(6″-p-coumaroyl-β- glucopyranoside) (Slimestadet al., 1995).

The ESI mass spectrum of compounds (5) showed [M-H]− at m/z583 corresponding to a molecular formulae C28H24O14 and character-ized as a flavone derivative on the basis of its UV absorption (λmax

366 nm), beside the appearance of the isolated singlet at δH ppm 6.09(3-H). Analysis of the 1H-NMR spectrum showed aromatic B-ring pro-ton appearing as two aromatic sets (A2B2) at δH ppm 8.11 and 6.93

OH

OH

OH

OH

O

O

O

Vitexin (7)

Nilocitin (3)Brevifolin carboxylic acid (2)

HO

HO

HO

HO

H

4 65

2

1

54a

4b

4

3

2

110

4 65

3 21

10a6a

6

7

893 OH

OH

OH OHO

O O

O O

O

O

OOH

OH

OH

OH

OH OH

OH

OH

HO

HO

HO

HO

HOOC

OH

HO

HO

HO

HO

HO HO

HO

7

6

45

8 12'

2'3'

87

6

5

4'

5'6'

1'

1'

7'

2'

3' 5'

6'

4'

4''

5''

6''

1''

7''

2''

3''1''

2'' 3''

3''

4''

6''

2''

5''

1''

1'

6'

5'

4'2'

3'

1

2

34

4''5''

6''

1'

1''

2''

2''

3''

3''

3'''

2'''

1'''

6'''

5'''

4''' 4''

4''

5'''6''' 7'''

5''6''

6'

5'

4'

3'

2

3

OH

OH

OH

OHOH

OH

OH

O

O

O

OO

O

HO

HO

HO HO

CO OC

O

O

O

OH

HO

HO

HO

HO

HO

HO HO

HO

HO

HO

7

6

45

8 1

7

6 45

8

2'

1'

1''

1'''

1'1''

2''

2''

2'

3''

3''

3'

4'

5'

6'2

33'''

2'''

1'''

6'''

5'''

4''' 4''

4''

5''

6''

5''6''

6'

5'

4'

3'

2

3

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

O

O

O

OO

O

O

O

O

O7

6 45

8

1'''1''

2''

2'3'

4'

5'

6'

1'

3''

4'' 6''

5''

7'''6'''5'''

4'''

3'''2'''

2

3

O

O

O

O

O

O

2,3-O-hexahydroxydiphenoyl

Kaempferol-3-O-(6"-p-coumaroyl-β- glucopyranose

(4)

-(αα/β)- glucopyranose (1)

2"-O-gallpyl vitexin (5) 2"-O-galloyl orientin (6)

Fig. 1. Chemical structures of compounds 1–7.

474 S.K. Gabr et al. / South African Journal of Botany 121 (2019) 470–477

denoting 2′, 6′ and 3′, 5′ respectively, a sharp downfield singlet inte-grated for two protons appeared at δH ppm 6.74 which denotes themagnetically equivalent 2 and 6 protons of a galloyl group. The aliphatic

Table 4Radical scavenging activity (IC50) for the isolated compounds of E × neillii leaf extract.

Test sample IC50 μg/ml

2,3 O-hexahydroxydiphenoyl-(α/β)-glucopyranose 9.73 ± 0.58Brevifolin carboxylic acid 5.12 ± 0.84Nilocitin 4.56 ± 1.84Kaempferol 3-O-(6″-p-coumaroyl-β- glucopyranoside 6.6 ± 0.952″-O-galloyl vitexin 3.72 ± 0.762″-O-galloyl orientin 1.85 ± 0.28Vitexin 10.5 ± 0.42Trolox 15.6 ± 1.63

Results are given in mean ± SD of three independent experiments.

proton signals are attributable to a β-D-glucopyranosyl moiety. Thedeshielding of 2″ H of the glucosyl residue which appeared at δH ppm5.50 (t) relative to the non galloylated analog vitexin indicates thatthe OH at this position was acylated. 13C-NMR confirmed the glucosyl

Table 5Docking score (Kcal/mol) of the isolated phenolics from the aqueousmethanolic extract ofE × neilliiwithin the active site of Heme oxygenase (1N3U) calculated by MOE.

Ligand Energy scores (Kcal/mol)

2, 3-O-hexahydroxydiphenoyl-(α/β)-glucopyranose −11.9851Brevifolin carboxylic acid −10.7644Nilocitin −16.3531Kaempferol-3-O-(6″-p-coumaroyl-β- glucopyranoside) −13.04872″-O-Galloyl vitexin −18.13352″-O-Galloyl orientin −18.6837Vitexin −10.8081

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

Brevifolin carboxylic acid

Nilocitin

Vitexin

Side chain acceptor/donor

Backbone acceptor/donor

Blue shadow represents ligand exposure

2¢¢-O-galloyl vitexin2¢¢-O-galloyl orientin

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

¢

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

475S.K. Gabr et al. / South African Journal of Botany 121 (2019) 470–477

attachment at C-8 by a significant downfield shift appearing at δC ppm104.60 compared with the non-glycosylated derivative.

The 13C-NMR downfield shift of the glucose carbon C 2″ (δC 73 ppm)in comparison with that of the corresponding carbon in free vitexin (δC70.85 ppm) (Agrawal et al., 1989) accompanied by the upfield shift ofthe anomeric carbon resonance (at δC 72 ppm) indicated that this posi-tion was substituted. Therefore compound (5) was identified as 2″-O-Galloyl vitexin.

Compound (6) showed [M-H]− at m/z 599 corresponding to a mo-lecular formula C28H24O15. Fragment at m/z 429 denoted the loss of agallic acid moiety (−170), while a fragment at m/z 309 [M-H-170-120] denotes the loss of a glucose moiety (Latté et al., 2002). 1H-NMRspectrum revealed similar features to compound (5) except for theABX-spin systems for the B-rings instead of the aromatic A2B2 previ-ously observed denoted by a doublet specific for 2′ at δH ppm 7.45 anda doublet of doublet denoting H-6′ at δH ppm 7.56. 1H and13C-NMRspectra displayed the typical signals for the spectra of flavones withC-8-hexosyl substituent (Latté et al., 2002; Rabe et al., 1994).Thus com-pound (6) was identified as 2″-O-Galloyl orientin.

Compound(7) appeared as the non-galloylated derivative of com-pound (5), showing typical signals of apigenin derivative (El-Masry

et al., 2010) with absence of H-8. The downfield shift of C-8 to δC ppm104.67 and C1″ of glucose at δC ppm 73.48 confirmed the attachmentof glucose moiety at C-8, therefore identified as vitexin (Latté et al.,2002). 1H and 13C- NMR data of compounds (4–7) are displayed inTables 1 and 2.

3.2. Oxygen radical scavenging capacity (ORAC) assay for isolatedcompounds

Due to the detrimental effects of oxidative stress in the different ail-ments and their role in the pathogenesis of cardiovascular disease, thisstudy was designed to verify the antioxidant activity of the isolatedcompounds using ORAC assay (Table 4) besides justifying this activitywith the molecular modeling to identify the binding interactions asshown in Table 5. Investigating the radical scavenging activity usingORAC assay resulted in the prevalence of 2″-O-galloyl orientin, whichshowed the highest antioxidant activity with IC50 1.85 μg/ml followedby 2″-O-galloyl vitexin with IC50 3.72 μg/ml while the lowest activitywas observed with vitexin (IC50 10.5 μg/ml).

Several reports showed that the radical scavenging capacities in-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,galloyl glucoses and ellagitannins (Fortes et al., 2015). In our study,the attachment of gallic acid to orientin, vitexin and its presence innilocitin justified a higher antioxidant capacity compared with thenon-galloylated compounds. Orientin higher scavenging activity com-pared to vitexin is attributed to the two hydroxyl groups in ring B(DimitrićMarković et al., 2017), while the high activity of brevifolin car-boxylic acid and 2,3-HHDP-glucopyranosemay be attributed to the car-boxylic group beside the phenolic hydroxyl groups (Chang et al., 2017;Karamac et al., 2005).

The role of phenolic compounds as radical scavengers has been wellstudied. However, another mechanism for antioxidants that has beenthe target formost of recent researches is compounds that can indirectlyupregulate endogenous antioxidant defenses, allowing profound anti-oxidant protection. Those compounds have long half-lives, and are un-likely to evoke pro-oxidant effects, therefore providing a moreefficient and long lasting response to oxidative stress. (Barbagalloet al., 2012). The results of the in-vitro antioxidant potential of the iso-lated compounds usingORAC assay showed pronounced activity as rad-ical scavengers. Based on this, amolecular docking studywas performedto document the mechanism of action of the isolated flavonoids andellagitannins as inducible for HO-1.

3.3. Molecular docking

HO-1 is an enzyme playing a vital role in the oxidative stress (hemeoxygenase). Upregulation of heme oxygenase-1 (HO-1), a phase II de-toxifying enzyme in endothelial cells, is considered to be helpful in car-diovascular disease. In addition, HO-1 could exert cytoprotective effectby preventing apoptosis (Lian et al., 2008). Understanding the hemeproteins structures and heme binding environment provide valuableguidelines in the design of novel antioxidants and anti-inflammatory(Choi and Alam, 1996). Molecular docking study of all compoundswas carried out on HO-1 (PDB code: 1N3U) to correlate the isolatedcompounds with the demonstrated activity through determining theinteractions of these compounds within the active site of HO-1.The co-crystallized ligand (Heme) was flexibly re-docked to verify thedocking protocol usingMMFF94 force field. The intermolecular interac-tions between the ligand and the target receptor was evaluated. A vali-dation for the ideal pose was performed by alignment of the X-raybioactive conformer, with the best-fitted pose of the same compoundfor the HO-1. The alignment showed good coincidence between them(RMSD = 0.7231 Å), indicating the validity of the selected pose. Theideal pose of each molecule was selected according to the energyscore and the best fitting to the active site. The predicted binding ener-gies of the compounds are listed in Table 5. Molecular docking study ofthe isolated compounds showed different binding interactions withinthe active site of HO-1 in addition to a correlation between the numberof phenolic (OH) groups and the binding score. The top five residueswith high relative frequencies reported in the substrate interactions ofHO-1 are cysteine (C), histidine (H), phenylalanine (F), methionine(M), and tyrosine (Y) (Li et al., 2011). In our study, the docking scoresfor the isolated compounds with HO-1 fall in the range of −10.8 to18.6 kcal/mol where all compounds interacted with Arg 183 and Lys18 except for 2″-O-Galloyl orientin achieving the highest radical scav-enging activity and showing the best docking score (−18.6837 Kcal/mol). 2″-O-Galloyl orientin formed a highly stable complex with theactive sites of the enzyme through forming four binding interactionswith residues, Arg 136 (two interactions), Met34 and Gly139. Thiswas followed by 2″-O-galloyl vitexin showing interactions with Arg136, Met 34 and Lys 18, while, nilocitin followed in activity showing in-teraction onlywithMet 34. The other compoundswere capable of bind-ingwith the active sitewith proper energy scores andwith the same keyamino acids which suggests identical binding mode. Stability of thecomplex (receptor–ligand) is increased by a higher number of hydrogenbonds as illustrated in Fig. 2. Binding to Met 34 represented a common

pattern for the most active compounds. It was reported that Met 34 (apolar thiol group) in the hydrophobic pocket of the heme structurehad a strong binding with electronegative moieties giving rise to a po-tent activity, in addition to a role in the stabilizing/destabilizing interac-tions (Rahman et al., 2012). This virtual screening study justified thehigher antioxidant activity of the tested compounds and their abilityto provide a direct and indirect antioxidant effects through their radicalscavenging activity and an induction of HO-1.

4. Conclusion

This study highlighted the importance of E × neillii that can repre-sent a promising effective candidate to combat oxidative stress due toits phenolic constituents. Additionally, our findings highlight that dur-ing the design of a novel antioxidant the conserved amino acids haveto be considered for enhancing the activity of the phytoconstituentsagainst HO-1.

Conflict of interest

The authors declare that there are no conflicts of interest.

Funding sources

This research did not receive any specific grant from funding agen-cies in the public, commercial, or not-for-profit sectors.

Acknowledgements

The authors are thankful for the Pharmaceutical Chemistry Depart-ment, Faculty of Pharmacy, MSA University for assisting in the molecu-lar docking study.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.sajb.2018.12.011.

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