Preparation and in vitro antioxidant activity of some novel flavone analogues bearing piperazine moiety

Preparation and in vitro antioxidant activity of some novel flavone analogues bearing piperazine moiety

Bioorganic Chemistry 95 (2020) 103513 Contents lists available at ScienceDirect Bioorganic Chemistry journal homepage:

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Bioorganic Chemistry 95 (2020) 103513

Contents lists available at ScienceDirect

Bioorganic Chemistry journal homepage:

Preparation and in vitro antioxidant activity of some novel flavone analogues bearing piperazine moiety


Paweł Berczyńskia, Aleksandra Kładnab, Oya Bozdağ Dündarc, Hatice Nehir Muratc, Elmas Sarıc, ⁎ Irena Kruka, Hassan Y. Aboul-Eneind, a

Institute of Physics, Faculty of Mechanical Engineering and Mechatronics, West Pomeranian University of Technology in Szczecin, Al. Piastów 48/49, 70-311 Szczecin, Poland b Department of History of Medicine and Medical Ethics, Pomeranian Medical University, Rybacka 1, 70-204 Szczecin, Poland c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, 06100 Tandoğan, Ankara, Turkey d Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Giza 12622, Egypt



Keywords: Piperazinyl flavone analogues Free radical inhibitory activity Antioxidant activity Chemiluminescence Electron spin resonance

Background: A series of eight new flavone derivatives containing a piperazine chain with different substitution were synthesized and their structures were determined. Methods: Their antiradical and antioxidant activities were evaluated using superoxide anion radical, hydroxyl radical, 2,2-diphenyl-1-picrylhydrazyl radical, 2,2′-azino-di(3-ethylbenzthiazoline sulphonate) radical cation (ABTS+%) scavenging (as measure total antioxidant status TAS), ferric reducing antioxidant power (TAC), and hydrogen peroxide decomposition. The antioxidant activities of the synthesized compounds were compared with standard antioxidants trolox, ascorbic acid, butylated hydroxytoluene (BHT) as positive controls, reference antibiotics (doxycycline, dicloxacillin), and medicinal plants (Menthae piperita, Cistus incanus). Chemiluminescence, spectrophotometry, electron spin resonance (ESR) spectroscopy in conjunction with 5,5dimethyl-1-pyrroline-1-oxide (DMPO) as the spin trap were the measurement techniques. Results: The results show that the synthesized compounds exhibit weak, albeit a wide spectrum of antiradical and antioxidant activities. The TAS values were measured as trolox equivalents, ranging from 209.6 ± 6.1 to 391.1 ± 8.2 µM TE/g; the TAC values were in ranges from 10.8 ± 0.5 to 49.5 ± 0.5 µM TE/g being higher than that of dicloxacillin (241.0 ± 16.5 and 9.73 ± 0.8 µM TE/g, respectively), but lower than ascorbic acid, BHT, doxycycline, and medicinal plants. Best antioxidant activities were found for the piperazinyl analogues with methoxy group on phenyl piperazine ring. Conclusion: We suggest that the synthesized compounds may be used as lead molecules for optimization of molecular structure to maximize the antioxidant potency.

1. Introduction The cellular damage caused by oxidative stress (OS) and its main contributing factors, i.e. excess of reactive oxygen species (ROS) (superoxide anion radical, O2¯ ; hydroxyl radical, HO%; and hydrogen peroxide, H2O2); and reactive nitrogen species (RNS), (nitric oxide, NO% and peroxynitrite, ONOO−) as well as too low activity of the cellular antioxidant systems to repair the cellular injury has been linked with

many chronic diseases and ageing [1–4]. During the past years the protective effects of antioxidants against the OS related pathophysiologies and antioxidant therapies have received increasing attention within medical field. Flavones are a group of naturally occurring compounds belonging to a large flavonoid family which has been found in all plants [5–7]. The group shows, among other, antioxidant activities against a wide spectrum of free radicals and non – radical ROS and strong protective effects against OS, offering reduction of the

Abbreviations: ABTS, 2,2′-azino-di(3-ethylbenzthiazoline sulphonate); ARP, antiradical power; BHT, butylated hydroxytoluene; CL, chemiluminescence; DMPO, 5,5dimethyl-1-pyrroline-1-oxide; DMSO, dimethyl sulfoxide; DPPH, 2,2-diphenyl-1-picrylhydrazyl; ESR, electron spin resonance; HO⋅, hydroxyl radical; H2O2, hydrogen peroxide; NMR, nuclear magnetic resonance; 1O2, singlet oxygen; O2¯· , superoxide anion radical; RNS, reactive nitrogen species; ROS, reactive oxygen species; SPF, substituted piperazinyl flavone; SV, stechiometric value; TAC, total antioxidant capacity; TAS, total antioxidant status ⁎ Corresponding author at: Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Giza 12622, Egypt. E-mail address: [email protected] (H.Y. Aboul-Enein). Received 17 September 2019; Received in revised form 16 November 2019; Accepted 16 December 2019 Available online 21 December 2019 0045-2068/ © 2019 Elsevier Inc. All rights reserved.

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civilization diseases risk [7–11]. They include anti – inflammatory, antimicrobial, antipathogenic, antiviral, anti-atherosclerotic, anticancer and cardioprotective effects. Evidence showed promotion of apoptosis of cancer cells such as prostate, lung and skin by natural and some of synthetic flavones [12]. The findings show the structure activity relationships: compounds having electron donating groups (eOH, eOCH3) on the phenyl ring favoured the antioxidant behaviour, while electron withdrawing atoms/groups (eF, eCl, eBr, eNO2) favoured the anti – inflammatory activity [13–15], and the presence of fluorine atom and SO2 group increases antioxidant and anti – inflammatory activities [13,16]. Several research laboratories in the world have focused on the synthesis effective plant derived compounds and their synthetic analogues as the potential therapeutics exhibiting antioxidant and anti – inflammatory activities. Importantly, sulfonyl, sulfonoamide [16], amino acids/peptides conjugated heterocycles [17] or benzisoxazole analogs [18] were developed as new candidates for less toxic drugs, among others. Similarly, piperazine and its derivatives are biologically active compounds displaying a broad range of activities, such as antioxidant and anti-inflammation, being recently used as anticancer and antibacterial agents [19–21]. An interesting research by Karthik and coworkers [22] showed novel compounds having piperazine in their structures (benzodioxane midst piperazine, BP and BP decorated chitosan silver nanoparticles) exhibit anti – inflammatory and anticancer properties among others. On the basis of these properties, incorporation of isoflavones and piperazines consists an important aspect of medicinal chemistry in developing of new antioxidant –based drugs. Based on the above facts and continuing our research program aimed in developing efficient synthesis of pharmacological useful new antioxidants, some new substituted piperazinyl flavones (SPF) were synthesized (Table 1) and their antioxidant activity in vitro was determined.

(Cistus incanus) collected in Turkey from Intenson Europe and (Menthae piperitae) collected in Poland were supplied from the local food market in Szczecin (Poland). 2.1.1. Preparation of the target compounds General procedure for preparation of SPF compounds studied is shown in Scheme 1. All synthesized compounds are novel. The general method which is known as Baker-Venkataraman method [23] was used to prepare 3′/4′-methyl flavone (Ia-b). The methyl group of the flavone was converted to bromomethyl (IIa-b) with N-bromosuccinimide (NBS) and a catalytic amount of benzoyl peroxide. Tert-butyl 4-(3 or 4-(4-oxo-4H-chromen-2-yl)benzyl)piperazine-1carboxylate (3B, 4B) was synthesized with 3′/4′-bromomethyl flavone IIa-b and tert-butyl piperazine-1-carboxylate, in the presence of Na2CO3/acetonitrile. The acidic hydrolysis of 3B, 4B provided corresponding piperazinylflavones 3H, 4H. Piperazinylflavone derivatives 4PP, 3PP, 4FCO, 3FCO, 43FMCO, 33FMCO, 4FBCO and 425 M were synthesized with 3H or 4H and appropriate substituted benzoyl halide or phenacylhalide in alkaline medium. Synthesis of 3′ (IIa)-4′ (IIb)-bromomethyl flavone. A mixture of N-bromosuccinimide (1.2 g, 6.72 mM) and 3′ (Ia)/4′-methyl flavone (Ib) (1.0 g, 4.2 mM) was dissolved in 70 mL of carbon tetrachloride and benzoyl peroxide (0.1 g) was added. The reaction mixture was refluxed for 7 h and filtered while it was still hot. The crude product was crystallized from ethylacetate: n-hexane. IIa m.p:154 °C (m.p.:137 °C[24]), IIb m.p.:158 °C (m.p.:139 °C [24]). General synthesis of tert-butyl 4-(3 (or 4)-(4-oxo-4H-chromen-2yl)benzyl)piperazine-1-carboxylate (3B-4B). A mixture of 3′/4′bromomethyl flavone IIa-b (0.50 g, 1.19 mM), tert-butyl piperazine-1carboxylate (0.29 g, 1.58 mM), Na2CO3 (0.16 g, 1.19 mM) and 10 mL acetonitrile was stirred at room temperature for 48 h. The reaction mixture was filtered, concentrated in vacuum, and then purified by column chromatography Silica gel 60 (230–400 mesh ASTM) using nhexane: ethylacetate (3: 1) as eluent.

2. Materials and methods 2.1. Reagents All the chemicals and reagents used in the study were of analytical grade and purchased from E. Merck (Darmstadt, Germany). The set of reagents for the quantitative determination of Total Antioxidant Status was obtained from Randox (UK). Commercial samples of herbal tea Tert-butyl 4-(3-(4-oxo-4H-chromen-2-yl)benzyl)piperazine-1carboxylate (3B). Yield: 0.48 g; 72.72%, m.p.: 113 °C (m.p.: 113 °C [24]).

Table 1 Piperazinyl flavone compounds.




















425 M




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Scheme 1. Synthesis of piperazinyl flavone compounds. tert-butyl 4-(4-(4-oxo-4H-chromen-2-yl)benzyl)piperazine-1carboxylate (4B). Yield: 0.49 g; 74.24%, m.p.: 138 °C (m.p.: 138 °C [24]).

mixture was evaporated in vacuum, water added and extracted with CHCl3 (3x50 mL). The organic layer was dried over anhydrous Na2SO4, evaporated to dryness and then purified by column chromatography Silica gel 60 (230–400 mesh ASTM) using dichloromethane: isopropanole (10: 1) as eluent. The pure viscous oil product was precipitated by adding ethylacetate. General synthesis of 3′/4′-(piperazin-1-ylmethyl)flavone (3H, 4H) [25]. A mixture of 3B-4B (0.48 g, 1.49 mM), 5 mL dioxane and 5 mL 4 M HCl was stirred at room temperature for 5 h. The solution of 10% Na2CO3 was added into the reaction mixture up to alkaline pH. This mixture was washed three times with CHCl3, and the combined organic extracts were washed with water. The organic phase was dried over Na2SO4, concentrated in vacuum, and then purified by column chromatography Silica gel 60 (230–400 mesh ASTM) using CHCl3: isopropanol: ammonium hydroxide solution (26–30%) (10:1:0.3) as eluent. 2-(4-((4-(benzo[d][1,3]dioxole-5-carbonyl)piperazin-1-yl) methyl)phenyl)-4H-chromen-4-one (4PP). Yield: 94%, m.p.: 141 °C. Spectroscopic analysis: 1H NMR (CDCl3, 400 MHz,δ, ppm): 2.48(broad, s, 4H, piperazine-CH2), 3.61(broad, s, 6H, piperazineCH2, fl-CH2), 5.98(s, 2H, O-CH2-O), 6.79–6,82 (m, 2H, 7′’,3-H), 6.89–6.92(m, 2H, 4′’,6′’-H), 7.42 (td, 1H, 6-H), 7.48–7.50 (m, 2H, 4′, 5′-H), 7.57(d, 1H, j8,7 = 8.40 Hz, 8-H), 7.69(td, 1H, 7-H), 7.88–7.89(m, 2H, 2′,6′-H), 8.23(d, 1H, j5,6 = 8.00 Hz, 5-H). MS (ESI + ) m/z (rel. Intensity): 469.4 (M + H, 100%); Anal. Calcd. for C28H24N2O4 C: 70.30, H: 5.51, N: 5.47%; found: C: 70.47, H: 5.54, N: 5.70%. 3′ – (piperazin-1-ylmethyl)flavone (3H). Yield: 0.30 g ; 81.08%, m.p.: 95 °C (m.p.: 95 °C [25]). 4′-(piperazin-1-ylmethyl)flavone (4H). Yield: 0.31 g ; 83.78%, m.p.: 100 °C (m.p.: 100 °C [25]). 2-(3-((4-(benzo[d][1,3]dioxole-5-carbonyl)piperazin-1-yl) methyl)phenyl)-4H-chromen-4-one (3PP). Yield: 89%, m.p.: 165 °C. Spectroscopic analysis: 1H NMR (CDCl3, 400 MHz,δ, ppm): 2.49(broad, s, 4H, piperazine-CH2), 3.61(broad, s, 4H, piperazineCH2), 3.63(s, 2H, fl-CH2), 5.97 (s, 2H, O-CH2-O), 6.79 (d, 1H, 7′’-H), 6.82(s, 1H, 3-H), 6.89–6.92(m, 2H, 4′’,6′’-H), 7.41 (td, 1H, 6-H), 7.45–7.48 (m, 2H, 4′, 5′-H), 7.57(d, 1H, j8,7 = 8.80 Hz, 8-H), 7.69(td, 1H, 7-H), 7.81–7.83(m, 1H, 6′-H), 7.88(s, 1H, 2′-H), 8.22(d, General synthesis of benzylsubstituted piperazin-1-ylmethyl flavones 4PP, 3PP, 4FCO, 3FCO, 43FMCO, 33FMCO, 4FBCO and 425 M. A mixture of 3′/4′-piperazinomethyl flavone 3H/4H (0.4 mM), substituted benzoylbromide (0.4 mM), Na2CO3 (0.4 mM) and 10 mL acetonitrile were stirred at room temperature for 12 h. The reaction 3

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1H, j5,6 = 8.00 Hz, 5-H). MS (ESI + ) m/z (rel. Intensity): 469.2 (M + H, 100%); Anal. Calcd. for C28H24N2O5-0,35C4H8O2 C: 70.72, H: 5.41, N: 5.61%; found: C: 70.86, H: 5.42, N: 5.77%.

CH2), 2.78 (s, 1H, piperazin-CH2), 2.85 (s, 1H, piperazin-CH2), 3.54 (s, 2H, fl-CH2), 3.68 (s, 3H, OCH3), 3.76 (s, 2H, COCH2), 3.77 (s, 3H, OCH3), 6.71 (s, 1H, 3-H), 6.80 (d, 1H, Ar-H), 6.92(dd, 1H, Ar-H), 7.21 (d, 1H, Ar-H), 7.32 (t, 1H, 6-H), 7.42 (d, 2H, jo = 8.40 Hz, 3′,5′-H), 7.47(d, 1H, j8,7 = 8.40 Hz, 8-H), 7.60 (td, 1H, 7-H), 7.78(d, 2H, jo = 8.40 Hz, 2′,6′-H), 8.12 (dd, 1H, j5,6 = 8.00 Hz, j5,7 = 1.20 Hz, 5H). MS (ESI + ) m/z (rel. Intensity): 441.6 (M + H, 100%); Anal. Calcd. for C30H30N2O5. 2-(4-((4-(4-fluorobenzoyl)piperazin-1-yl)methyl)phenyl)-4Hchromen-4-one (4FCO). Yield: 88%, m.p.: 164 °C. Spectroscopic analysis: 1H NMR (CDCl3, 400 MHz,δ, ppm): 2.49 (broad s, 4H, piperazine-CH2), 3.48 (broad s, 2H, piperazine-CH2), 3.62 (s, 2H, flCH2), 3.78 (broad s, 2H, piperazine-CH2), 6.82 (s, 1H, 3-H), 7,06–7,10 (m, 2H, Ar-H), 7,39–7,44 (m, 3H, Ar-H, 6-H), 7.49 (d, 2H, jo = 8.40 Hz, 3′,5′-H), 7.56 (d, 1H, j8,7 = 8.80 Hz, 8-H), 7.70 (td, 1H, 7-H), 7.89 (d, 2H, jo = 8.0 Hz, 2′,6′-H), 8.23 (d,1H, j5,6 = 8 Hz, 5-H). MS (ESI + ) m/z (rel. Intensity): 443,2 (M + H, 100%); Anal. Calcd. for C27H23FN2O30,2H2O C: 72.69, H: 5.29, N: 6.28%; found: C: 72.65, H: 5.56, N: 6.11%.

2.1.2. Procedures for preparation free radicals The superoxide anion radical (O2¯ ) was prepared from KO2 according to the procedure of Valentine et al.[26]. Initially, 60 mg of 18-crown-6 ether was dissolved in 10 mL dry DMSO and 7 mg of KO2 was added quickly to avoid contact with the air humidity. The reaction mixture was stirred for 1 h to give a pale yellow solution of 10 mM superoxide radical which was stable at room temperature. The radical concentration was measured with the UV–VIS spectrophotometer (λmax = 251 nm, ε = 2686 ± 29 M−1 cm−1). For the experiments, O2¯ was used as a 1 mM solution in DMSO. Hydroxyl radicals (HO%) were generated in the Fenton reaction (H2O2 + Fe(II) → HO. + Fe(III) + HO−) [27,28]. 2-(3-((4-(4-fluorobenzoyl)piperazin-1-yl)methyl)phenyl)-4Hchromen-4-one (3FCO). Yield: 87%, m.p.: 75 °C. Spectroscopic analysis: 1 H NMR (CDCl3, 400 MHz,δ, ppm): 2.55(broad s, 4H, piperazine-CH2), 3.49(broad s, 2H, piperazine-CH2), 3.65(s, 2H, fl-CH2), 3.81(broad s, 2H, piperazine- CH2), 6.86(s, 1H, 3-H), 7.08–7.12(m, 2H, Ar-H), 7,42–7,53(m ,5H, Ar-H, 6, 4′, 5′H), 7.61(d, 1H, j8,7 = 8.40 Hz, 8-H), 7.73(td, 1H, 7-H), 7.84–7.87(m, 1H, 6′-H), 7.91(s, 1H, 2′-H), 8.25(dd, 1H, j5,6 = 8.00 Hz, j5,7 = 1.20 Hz, 5-H). MS (ESI + ) m/z (rel. Intensity): 443,2 (M + H, 100%); Anal. Calcd. for C27H23FN2O3-1,2H2O C: 69.87, H: 5.51, N: 6.04%; found: C: 69.79, H: 5.20, N: 6.09%.

2.1.3. Preparation of herbal teas infusions 1.5 g of the commercial Menthae piperitae and Cistus incanus were dipped separately into 100 mL of freshly boiled redistilled water in a beaker, shaken and allowed to stand for 10 min. Next tea infusions were allowed to cool to room temperature and filtered through a Whatman filter paper. 2-(4-((4-(4-(triflorometoksy)benzoil)piperazin-1-il)methyl) fenyl)-4H-kromen-4-on (43FMCO). Yield: 95%, m.p.: 143 °C. Spectroscopic analysis: 1H NMR (CDCl3, 400 MHz,δ, ppm): 2.45(broad s, 2H, piperazine- CH2), 2.55(broad s, 2H, piperazineCH2), 3.45(broad s, 2H, piperazine-CH2), 3.62(s, 2H, fl-CH2), 3.80(broad s, 2H, piperazine-CH2), 6.82(s, 1H, 3-H), 7,24(d,2H,Ar-H) 7,40–7,46(m,3H,Ar-H,6-H) 7.49(d, 2H, jo = 8.0 Hz, 3′,5′-H), 7.56(d, 1H, j8,7 = 8.80 Hz, 8- H), 7.70(td, 1H, 7-H), 7.89(d, 2H, jo = 8.0 Hz, 2′,6′-H), 8.23(dd,1H, j5,6 = 8.00 Hz, j5,7 = 1.20 Hz,5-H). MS (ESI + ) m/z (rel. Intensity): 509,3 (M + H, 100%); Anal. Calcd. for C28H23FN2O4 C: 66.14, H: 4.73, N: 5.64%; found: C: 66.14, H: 4.73, N: 5.64%.

2.2. Apparatus Melting points of the compounds were measured on an Electrothermal 9100 type apparatus (Electrothermal Engineering, Essex, UK) and uncorrected. All instrumental analyses were performed in Central Laboratory of Faculty of Pharmacy of Ankara University. 1H NMR spectra were determined with a VARIAN Mercury 400 FT-NMR spectrometer (Varian Inc, Palo Alto, CA, USA) in CDCl3 and DMSO‑d6. All chemical shifts were reported as δ (ppm) values. Mass spectra were recorded on Waters Micromass ZQ (Waters Corporation, Milford, MA, USA) by using ESI (+) method. Elementary analyses were performed on a Leco CHNS 932 analyzer (Leco, St. Joseph, USA) and satisfactory results ± 0.4% of calculated values (C, H, N) were obtained. For the chromatographic analysis Merck Silica Gel 60 (230–400 mesh ASTM) was used. The chemiluminescence (CL) measurements were performed using a luminometer equipped with an EMI 9553Q photomultiplier (Photek, East Sussex, UK) with a S20 cathode sensitive in the range 200–800 nm. A thermostated glass cuvette placed in a light-tight camera was exhausted after reaction using a B-169 vacuum system (Büchi, Flawill, Switzerland). The measurements were carried out at room temperature. Spectrophotometric measurements were performed using a UV/VIS spectrophotometer equipped with a thermostat bath (Jasco V-550). Electron spin resonance (ESR) spectra were recorded with a standard X-band spectrometer operating at 9.3 GHz with a 100 kHz modulation of the steady magnetic field. Samples were introduced into the cavity in a quartz cuvette with an optical path length of 0.25 mm and recorded after 1 min from the start of a reaction and analyzed every one minute. The majority of the data are presented as a mean ± standard error of the mean. With the exception of the total antioxidant status (TAS) evaluation, the remaining experiments were carried out at room temperature. Statistical evaluation was carried out using the basic statistics and regression analyses using the statistical package STATISTICA 6.0 2002 (StatSoft Polska, Kraków). A p-value < 0.05 was considered to be significant. 2-(3-((4-(4-(triflorometoksy)benzoil)piperazin-1-il)methyl) fenyl)-4H-kromen-4-on (33FMCO). Yield: 93%, m.p.: 138 °C. Spectroscopic analysis: 1H NMR (CDCl3, 400 MHz,δ, ppm): 2.45(broad s, 2H, piperazine-CH2), 2.57(broad s, 2H, piperazine-CH2), 3.46(broad s, 2H, piperazine-CH2), 3.64(s, 2H, fl-CH2), 3.81(broad s, 2H, piperazine-CH2), 6.84(s, 1H, 3-H), 7,24(d, 2H, Ar-H), 7.41–7.50(m, 5H, Ar,4′,5′,6-H) 7.58(d, 1H, j8,7 = 8.0 Hz, 8-H), 7.71(td, 1H, 7-H), 7.84(d, 1H, 6′-H), 7.90(s, 1H, 2′-H), 8.23(d, 1H, j5,6 = 8.00 Hz, 5-H). MS (ESI + ) m/z (rel. Intensity): 509 (M + H, 100%); Anal. Calcd. for C28H23FN2O4 C: 65.38, H: 4.85, N: 5.15%; found: C: 65.08, H: 4.76, N: 5.45%. 2-(4-((4-(2-(4-fluorophenyl)-2-oxoethyl)piperazin-1-yl)methyl) phenyl)-4H-chromen-4-one (4FBCO). Yield: 84.11%, m.p.: 123 °C. Spectroscopic analysis: 1H NMR (CDCl3, 400 MHz,δ, ppm): 2.62 (broad s, 4H, piperazine-CH2), 3.67 (broad s, 4H, piperazineCH2), 3.63 (s, 2H, fl-CH2), 3.79 (s, 2H, COCH2), 6.82 (s, 1H, 3-H), 7.09–7.14 (m, 2H, Ar-H) , 7.42 (td, 1H, 6-H),7.51 (d, 2H, jo = 7.60 Hz, 3′,5′-H), 7.57 (d, 1H, j8,7 = 8.80 Hz, 8-H), 7.710 (td, 1H, 7-H), 7.89 (d, 2H, jo = 8.00 Hz, 2′,6′-H), 8.02–8.06 (m, 2H, Ar-H), 8.23 (dd,1H, j5,6 = 8.00 Hz, j5,7 = 1,60 Hz,5-H). MS (ESI + ) m/z (rel. Intensity): 457.5 (M + H, 100%); Anal. Calcd. for C28H25FN2O3. 2-(4-((4-(2-(2,5-dimethoxyphenyl)-2-oxoethyl)piperazin-1-yl) methyl)phenyl)-4H chromen-4-one (425 M). Yield: 56.22%, m.p.: 128 °C. Spectroscopic analysis: 1H NMR (CDCl3, 400 MHz,δ, ppm): 2.53 (broad s, 3H, piperazin-CH2), 2.61 (broad s, 3H, piperazin4

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2.3. Antioxidant assays

were plotted for the compound tested concentration in the range of 0.125 – 1.25 mM and the percentage of DPPH% remaining at the steady state was read. Then, the percentage of DPPH% was plotted as function of the molar ratio of the tested compound to DPPH%, according to the procedure of Brand – Williams et al.[32]. From the graph, the Efficient Concentration (EC50) value specifying the concentration of an antioxidant sample that causes a 50% decrease of ESR signal amplitude compared with the amplitude of the untreated reference reaction (M antioxidant/M DPPH%) was determined. Also, the antiradical power (ARP) defined as 1/EC50 (the value directly proportional to antioxidant activity), stoichiometric value (SV = 2EC50) describing the theoretical efficient concentration of antioxidant necessary to scavenge 100% of the initial DPPH%, and the number of reduced DPPH radicals calculated as 1/SV were obtained for a few tested compounds and the reference compounds. Ascorbic acid and BHT were used as the positive controls.

2.3.1. Reactivity of SPF towards the superoxide anion radical Reactivity of synthesized compounds towards O2¯ was examined in DMSO in the range from 0.1 to 0.5 mM using 1 mM O2¯ . The resulting CL signal from the O2¯ solution (blank) and that influenced by the tested compounds were recorded as the kinetic curves of the CL decay. Then the CL intensity sums were calculated as the area under these curves for 5 min duration. The effects were expressed as the enhancing ratio Q (%) of the light sum as follows: Q (%) = [(ΣI − ΣI0)/ΣI0] × 100%, where ΣI0 is the integrated light intensity measured in the absence of SPF compound but in the presence of 0.5 mL of DMSO, and ΣI is the sum measured in the presence of the compound. 2.3.2. Hydrogen peroxide scavenging assay The H2O2 scavenging ability was measured by monitoring the response of CL accompanying H2O2 – induced oxidation of luminol using a previously described by Tao et al. assay [29]. Reaction mixture contained the following reagents at the given final concentrations: 100 μM of luminol, 5 mM H2O2, and phosphate buffer pH 7.0. The reaction was initiated by an addition of H2O2 , and 15 s after the start of the light emission and reaching maximal intensity 0.5 mM of a test compound dissolved in DMSO or the same volume of alone DMSO was added to the reaction mixture. Effects were expressed as the percentage of the H2O2 – induced oxidation of luminol or the standard compound (ascorbic acid) Q (%) = [(ΣI0 − ΣI)/ΣI0] × 100%, where ΣI0 is the CL sum of the control, and ΣI is the CL sum in the presence of the tested compounds or the standard.

2.3.5. Determination of total antioxidant status (TAS) Total antioxidant activity of the examined compounds was measured using the TAS RANDOX kit (Cat. No.NX2332, Randox Laboratories Ltd., Co. Antrim, UK), in accordance with the instruction. The method is based on reduction of 2,2′-azino-di(3-ethylbenzthiazoline sulfonate) radical cation (ABTS+%) to an extent dependent on the antioxidant potential [33]. This cation radical is formed in the reaction of one-electron oxidation of ABTS during incubation with a peroxidase (metmyoglobin) and H2O2. A degree of suppression of the radical cation was compared to the antioxidant activity of standard concentrations of Trolox and expressed in the units of µM Trolox equivalent (TE) per gram of the tested compound dry mass. Ascorbic acid, BHT, dicloxacillin, doxycycline, menthae piperitae, and cistus incanus were used as the positive controls.

2.3.3. Hydroxyl radical scavenging assay The HO% scavenging activity was evaluated by using the Fenton reaction and DMPO as a spin-trap, according to a previously described method [30]. The method allows to detect a short-lived HO% forming with the spin-trap a more stable free radical as DMPO ─ %OH adduct. Reaction mixture contained the following reagents at the final concentrations: 10 mM sodium trifloroacetate (pH 6.15), 25 mM DMPO, 0.5 mM H2O2, 0.625 mM ammonium-ferrous sulfate, and the tested compounds (dissolved in DMSO due to their insolubility in water). The tested compounds were added before the Fe ion addition, which starts the Fenton reaction. After mixing reagents, the mixture was transferred to an ESR spectrometry cell and one min after ESR spectrum was recorded. The known antioxidants BHT and thiourea were used as the positive controls. The results were expressed as a percentage of the signal inhibition using a formule: Q (%) = [(H0 − H)/H0] × 100%, where H0 and H represent the relative height of the spin – adduct from the blank (DMSO) and in the presence of a sample of the tested compound dissolved in an appropriate amount of DMSO, respectively. Measurement conditions of ESR were: field weep 330.5–340.5 mT, field modulation with 0.5 mT, time constant 0.3 s, microwave power 20 mW, and receiver gain 2x104.

2.3.6. Ferric reducing ability measurements (TAC) The reducing activity was evaluated by monitoring the reduction of the Fe(III) – ferrozine agent to stable Fe(II)- ferrozine complex according to a described procedure by Berker et al. [34]. The ferric-ferrozine complex contained of the following reagents at the indicated final concentrations: 2 mM Fe(III) and 10 mM ferrozine. The complex was prepared as follows: the water solution of 0.024 g of NH4 Fe (SO4)2·12H2O after an addition of 1 mL HCl (1 M) was mixed with a separate prepared aqueous solution of 0.123 g ferrozine. The mixture was diluted to 25 mL with distilled water. Then, 0.5 mL of a tested compound dissolved in DMSO was mixed with 1.5 mL of ferric-ferrozine solution and 2 mL of acetate buffer pH 5.5 (0.2 M). To obtain the final required volume 4.5 mL, 0.5 mL of water was added. The mixture was shaken vigorously and allowed to stand at 25 °C for 1 h. The spectrophotometric assays were performed at the absorption wavelength 562 nm. Ascorbic acid, BHT, dicloxacillin, doxycycline, menthae piperitae and cistus incanus were used as the positive controls. The reducing activity was expessed in the units of µM Trolox Equivalent (TE) per gram of the tested compound dry mass.


3. Results and discussion

2.3.4. 2,2 - diphenyl – 1-picrylhydrazyl radical scavenging activity The DPPH% scavenging activity was measured using a previously described methodology by Nanjo et al. [31] with the small modification, i.e. the solvent mixture DMSO:C2H5OH(1:3) was used instead of ethanol. The method is based on spectroscopic monitoring changes of a stabile DPPH free radical in the presence of antioxidants. The tested compounds dissolved in DMSO at various concentrations were mixed with ethanolic solution of DPPH (0.25 mM final concentration) and a decrease in ESR spectrum amplitude was detected. The decrease in a spectrum amplitude was monitored until the reaction reached a plateau. The scavenging activity of the tested compounds towards DPPH% was calculated using the following equation: Q (%) = [(H0 – H)/ H0] × 100%, where H0 is the relative height of the third peak in the ESR spectrum in the absence of a test compound, H is the relative height of the third peak in the presence of the compound. The reaction kinetics

The synthetic routes for the newly synthesized compounds are presented in the Scheme 1 and described in the Materials and Methods. The structure of the synthesized compounds was elucidated by elementary analysis, 1H NMR, and mass spectral data. All spectral data were in accordance with assumed structures. In 1H NMR spectra, flavone protons were observed between 6.79 and 8.25 ppm (see Supplementary data). Mass analysis of compounds was performed by using ESI (+) method. All the compounds have M + H ion peaks. The series of a novel group of piperazinyl flavones were evaluated as possible antioxidant agents by performing various tests, like reactivity towards: O2¯ , HO%, DPPH%, ABTS%+, and H2O2. Additionally, the ability to reduce Fe(III) ion to Fe(II) ion, which is a significant indicator of antioxidant potency, was evaluated using the Fe(II) – ferrozine complex 5

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[34]. The results are compared with those obtained for several standard antioxidants, antibiotics and medicinal plants. The O2¯ , H2O2 and HO% are produced during endogenous metabolic processes in the human body as the normal product of one – electron reduction of molecular oxygen (O2) and also are formed from external sources, like environment pollution or radiation [1,35]. The O2¯ radical is known as precursor of more chemically active HO% and singlet oxygen (1O2) formed in biological system [36,37]. The oxygen free radicals are short – lived and highly reactive; they can behave as oxidants or reductants and cause damage of the cellular macromolecules, like nucleic acids, lipids, proteins, and carbohydrates [1,3]. An imbalance between formation of ROS and antioxidant defenses with a predominance of oxidation processes is known in literature as oxidative stress (OS) that has been reported to contribute to several serious diseases, including cancer and premature aging [38,39]. Compounds showing antioxidant activity act, among other, as direct radical scavengers, hydrogen or electron donors or peroxide decomposers. We found previously that 1O2 is responsible for CL accompanying the O2¯ /DMSO system detecting four emission bands with maxima at 480 nm, 580 nm, 640 nm, and 700 nm which can result from radiative deactivation of 1O2 – dimoles (1O2)2 to the ground state (3O2) [40]. In agreement with reactivity of O2¯ in DMSO [41], the increased light emission (Fig. 1) in the presence of SPF compounds may be due to the proton transfer to the anion radical followed by hydroperoxyl radical (HO2· ) formation

FSP(H) + O¯2·

Fig. 2. Hydrogen peroxide and hydroxyl radical inhibitory activity at concentration 0.5 mM and 1.25 mM of piperazinyl flavones, respectively. Data are represented as mean ± S.D.(n = 3). The chemiluminescence and electron spin resonance signals were measured as explained under Materials and Methods. Denotations of the compounds are shown in Table 1.

groups in structure of the tested compounds. In addition, the compounds having substitution R at position 4′ (Table 1) were found to be more reactive than those with substitution at position 3′. This suggests that the stabilization of the products formed from FSP transformation is higher for R attached to the chromone skeleton at position 4′. This suggestion agrees with Phosrithong et al.’ findings [43] that the chromone skeleton plays only role of a stabilizer during the hydrogen or/ and electron transfer to a free radical. The behaviour of SPF compounds reminds that of superoxide dismutase, an enzyme which transforms O2¯ to H2O2. The ability of SPF compounds to scavenge hydrogen peroxide and hydroxyl radical is presented in Fig. 2. The H2O2 scavenging ability of SPF compounds was detected using a previously described chemiluminescent assay based on the detection of light emitted during H2O2 – induced oxidation of luminol [44]. The overall range of effective H2O2 inhibition was found to be 23.5 – 98.0% (425 M > 4FBCO > 4FCO > 33FMCO > 3FCO > 43FMCO > 4PP ≥ 3PP). The positive control ascorbic acid exhibited 40.4% quenching of CL at the same concentration as the all compounds (0.5 mM) except for 425 M compound which was the most effective derivative, providing 98% inhibition at concentration of 0.1 mM. Compounds 425 M, 4FBCO were more effective than ascorbic acid. The structure of these two piperazinyl flavone compounds have a methylene bridge between piperazine ring and benzoyl group. In this reaction O2¯ plays a key role in the generation of electronically excited 3- aminophthalate of an emitter of the light emission observed at 426 nm [29]. The efficiency of the antioxidant action relies on the rapidity of the O2¯ removing from the reaction mixture followed by the decreased formation of the CL emitter. By comparing Fig. 2 with Fig. 1, we assume the same hierarchic order of antioxidant potency. The results from the H2O2/luminol assay show, once again, the importance of a number of methoxy groups and the 4′ substitution for the antioxidant activity of SPF compounds. The ability of SPF compounds to scavenge the HO radical was studied by ESR spectrometry using DMPO as the spin trap. This is the routinely applied technique for monitoring the short – lived oxygen free radicals. The rate constant for reaction of HO%, formed in the Fenton reaction using ethanol as the solvent, with DMPO spin trap is very high (2.1 × 109 M−1s−1) [30]. The radical trapped by DMPO gives a spin – adduct DMPO-OH which exhibits a characteristic ESR spectrum containing four – splitted lines with an intensity ratio 1:2:2:1 and a hyperfine – splitting constant of aN =aN = 14.9 G , as shown in Fig. 2. The

FSP + HO·2

The self reaction of HO2· leads to the 1O2 formation [42]


1O 2

+ H2 O2

Superoxide radical interaction of the synthesized compounds was assessed using CL accompanying oxidation of the radical in DMSO due to their solubility in this solvent. All determinations are carried out in triplicate, and the results are expressed as enhancing ratio of CL sum detected from the O2¯ /DMSO system in the presence of SPF compounds. The obtained results are shown in Fig. 1. All the tested compounds showed a strong significant (p < 0.001) 2.4 – 17.2 – fold increase in the CL sum at concentration of 0.1 mM being a dose – dependent. Among considered structures, compounds (425 M, 4FBCO, 4PP and 3PP) bearing two methoxy groups, fluorine atom or methylenedioxy substituents were the most efficient as enhancers of light emission. The results seem to be largely affected by the number of methoxy

Fig. 1. Effect of piperazinyl flavone derivatives on the chemiluminescence recorded from the superoxide anion radical/DMSO reaction. Chemiluminescence sums were measured as explained under Materials and Methods. Data are shown as mean ± S.D. (n = 3). Denotations of the compounds are shown in Table 1. 6

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The ABTS+% scavenging is a widely used an antioxidant capacity assay classified as an electron – transfer based assay (TAS) [51]. The radical cation scavenging activity of the test compounds, measured by using the Randox test, is listed in Table 2. The data are presented as µM of trolox per 1 g of a sample to compare the reactivity of the synthesized compounds with the standard scavengers, ascorbic acid and BHT. Previously we found that some antibiotics belonging to penicillin [52] and tetracyclin [53] groups exhibited antioxidant activity in different in vitro assays. In view of these facts it seemed interesting to compare the antioxidant properties of SPF compounds with those of dicloxacillin and doxycycline as well as the herbal tea infusions Menthae piperitae and Cistus incanus known with their antioxidant ability. Antioxidant capacity of the test compounds as measured by the ABTS+% method followed the order: Ascorbic acid > Cistus incanus > Menthae piperitea > BHT > Doxycycline > 425 M > 4PP > 3PP > 4FBCO > 43FMCO > 4FCO > 3FCO > Dicloxacillin > 33FMCO. The synthesized compounds showed considerable less antiradical capacity than the positive controls, medicinal plants, and doxycycline. Although, the majority of the synthesized compounds were significantly stronger than dicloxacillin. The experiment provided confirmation of our earlier findings [25] about an importance of OCH3 group and fluorine atom present in the substituent R (Table 1) for the antioxidant property. Both the groups can donate an electron to free radicals [54]. The low TAS values observed for SPF compounds suggest an electron donor mechanism of antioxidant activity. Ferric ion reducing antioxidant capacity of the synthesized compounds was evaluated in the TAC assay based on the Fe(III) to Fe(II) reduction [34]. The assay uses antioxidants as Fe(III) ion reductants. It has been reported that the ferric reducing potency is correlated with a compound electron - donating antioxidant power [55]. This ability was monitored using transformation of the ferric – ferrozine complex to a ferrous/ferrozine form by measuring the change in the absorption at 562 nm. The method is simple, convenient and able to measure the total antioxidant capacity of pure substances and plant infusions [34]. The TAC values for the tested compounds were expressed in the units of µM TE/g (Table 2). These values for SPF compounds ranged from 10.8 µM TE/g to 49.5 µM TE/g, showing little Fe(III) reducing ability; they were only higher than that detected for dicloxacillin (TAC = 9.73 ± 0.8) and much more lower than those measured for ascorbic acid, BHT, doxycycline, and medicinal plant infusions, under the same reaction conditions. The total antioxidant status of the synthesized compounds evaluated by ABTS radical scavenging activity was highly correlated with that determined using the Fe(III) → Fe(II) transformation assay (r = 0.89069, p < 0.003). This finding confirms that the single – electron transfer is the main mechanism through which the synthesized compounds may express their antioxidant potential. The synthesized compounds in this paper become distinct by substitute of benzene ring of the R substituent in piperazine ring and a linkage (3′ or 4′ positions) of piperazine to flavonoid skeleton. Therefore, the differences of their antioxidant potency are discussed correlating with the newly synthesized structures. The main structure feature responsible for the free radical scavenging and antioxidant activity is the presence of fluorine atom, methoxy group on benzoyl group, the potential electron – donating methylenedioxy substituent [43,56] and a methylene bridge beteen piperazine ring a bezoyl group. The methylene bridge provides free rotation between these two structures. In turn, stereo chemical factors linked with 3′ or 4′ position might be the cause for the differences in antioxidant activity due to the stability of the product. The presented findings show that in the all applied methods for evaluation of antiradical and antioxidant activities, the highest activity was demonstrated by compound 425 M. It seems that this result is due to the presence of two – OCH3 groups in the agent structure. The presence of two –CH3 groups may stronger increase the electron density in benzene ring via electron – donating than the presence of one group (33FMCO, 43FMCO). The differences in the

parameters we obtained for HO% trapping are consistent with those reported by other authors [45,46]. The tested compounds displayed poor HO radical scavenging activity, ranging from 9% to 32% in comparison with cistus incanus (44.2%, data not shown). However thiourea, exhibiting a high reactivity towards HO% (~109 M−1s−1) [47], showed comparable radical scavenging activity (30%) as 425 M compound (32%). The percentage of HO%- scavenging activity shown by SPF compounds refers to the highest concentration of the synthesized compounds, due to their precipitation observed at higher concentrations. We observed that the presence of DMSO (3.5 M) in the Fenton reaction needed to dissolve a SPF compound, reduced the DMPO -%OH signal magnitude by 73%. The concentration of DMSO significantly exceeded the DMPO concentration (25 mM). In addition, its rate constant for HO% trapping (7 × 109 M−1s−1) [48] was greater than the DMPO rate constant (3.4 × 109 M−1s−1 [30]. This suggests that most of HO% was removed by DMSO. Indeed, we noticed the formation of a new spectrum similar to the DMPO -%CH3 adduct due to the CH3% derived from DMSO [48] (data not shown). Among a number of method applied to determine the antioxidant ability to trap free radicals, the DPPH method is most commonly used due to its simplicity and reliability. The DPPH free radical decrease in the presence of an antioxidant agent was monitored using ESR spectroscopy. Owing to the DPPH radical’s ability to receive hydrogen or an electron from an antioxidant agent, the radical become a stable diamagnetic molecule. The scavenging effects of SPF compounds are shown in Table 2. Two positive controls, ascorbic acid and BHT are also included. Only two, 4FBCO and 425 M, of 8 synthesized compounds reduced DPPH% in a dose – dependent manner, presenting much weaker antiradical activity, ARP, than an endogenic antioxidant ascorbic acid, which presented an ARP value of 4.08 or the synthetic antioxidant BHT (ARP = 2.86). These values for the positive controls agree with those found by other authors [49,50]. The remaining tested agents provided from 13.2% to 18.2% the radical scavenging effect. Table 2 Antioxidant activity of the tested piperazinyl flavone compounds, positive controls, reference antibiotics and medicinal plants. Compound

3PP 4PP 3FCO 4FCO 33FMCO 43FMCO 4FBCO 425 M Positive controls Ascorbic acid BHT Reference antibiotcs Dicloxacillin Doxycycline Medicinal plants Menthae piperitae Cistus incanus

DPPH radical scavenging activity





[µM TE/g]

[µM TE/g]

16.0 ± 18.2 ± 13.2 ± 14.8 ± 13.7 ± 15.3 ± 0.244 0.435

2.5* 3.1* 2.4* 2.3* 3.0* 3.3* 8.2 4.6

0.122 0.217

264.5 308.6 243.2 255.1 209.6 259.4 260.4 392.1

23.8 20.8 15.2 17.5 10.8 15.0 13.5 49.5

4.08 2.86

0.49 0.70

2.04 1.43

– – –

Number of reduced DPPH.

± ± ± ± ± ± ± ±

7.7 7.9 6.7 7.1 6.1 6.9 6.5 8.2

± ± ± ± ± ± ± ±

0.3 0.4 0.3 0.3 0.5 0.5 0.6 0.5

6006 ± 196.8 1304 ± 43.2 241 ± 16.5

5009 ± 98.7 217 ± 5.3 9.73 ± 0.8

539 ± 28.3

428 ± 27.1

1341 ± 39.7 1538 ± 42.5

470 ± 27.8 876 ± 37.3

* Scavenging effect (mean%) at the highest tested concentration (1.25 mM). Time of steady state of the synthesized compounds in the DPPH assay was 30 min. ARP – antiradical power; SV – stechiometric value; TAS – total antioxidant status; TAC – total ferric reducing power; . BHT – butylated hydroxytoluene. Data are expressed as mean ± standard deviation of n = 3 determinations. Denotations of the synthesized compounds are listed in Fig. 1. 7

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antiradical power observed among the particular assays result from multiple mechanisms and different reaction conditions, as well as from different oxidant behavior of the test compounds. It is noteworthy that the synthesized compounds showed broad spectrum of antioxidant activity in both in hydrophobic medium, H2O/DMSO, and H2O/DMSO/ C2H5OH solutions, being scavengers of ROS and DPPH and ABTS+ radicals.

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4. Conclusion The findings allow us to judge that the synthesized compounds have the potential as antioxidants. A series of novel piperazinyl flavone compounds was synthesized and their structure was elucidated by elementary analysis, chromatography, 1H NMR spectroscopy, and mass spectral data as response to the pharmacy industry’s demand for new non – toxic providing antioxidant defense against ROS. The results agree with the assigned structures. The compounds were in vitro tested for their ability to scavenge free radicals and antioxidant activity. From the performed evaluation results that all the compounds showed weak free radicals scavenging and the ferric ion reducing potentials activities when compared to the standard controls. Compound 425 M demonstrated the highest significant inhibitory effect in all six assays used. The findings showed that difference in the chemical structure of the substituent R reflects mainly to the observed antioxidant power and scavenging of free radicals. It is apparent that the presence of methoxy groups on benzene ring of the substituent R (compound 425 M) linked with piperazine ring is essential for the antioxidant and free radical inhibiting properties. The findings allow us to judge that the synthesized compounds may be used as lead molecules for optimization of molecular structure to maximize the antioxidant potency. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgements This work was supported by Research Organization of Ankara University, Ankara, Turkey (No: 12B3336003) and keeping the research potential in frame of statutory activity of West Pomeranian University of Technology in Szczecin, Szczecin, Poland (No: 518-060123161-03/ 18). Appendix A. Supplementary material Supplementary data to this article can be found online at https:// References [1] V. Valko, D. Leibfritz, J. Moncol, M.T.D. Cronin, M. Mazur, J. Telser, Free radicals and antioxidants in normal physiological functions and human disease, Int. J Biochem. Cell Biol. 39 (2007) 44–84. [2] F.S. Pala, H. Gürkan, The role of free radicals in ethiopathogenesis of diseases, Adv. Mol. Biol. 1 (2008) 1–9. [3] S. Reuter, S.C. Gupta, M.M. Chaturvedi, B.B. Aggarwal, Oxidative stress, inflammation, and cancer: how are they linked? Free Radic. Biol. Med. 49 (2010) 1603–1616. [4] M. Venza, M. Visalli, C. Beninati, G.V. De Gaetano, D. Teti, I. Venza, Cellular mechanisms of oxidative stress and action in melanoma. Oxid. Med. Cell Longev., (2015) ID 481782, [5] X.-F. Guo, Y.-D. Yue, F. Tang, J. Wang, X. Yao, Antioxidant properties of major flavonoids and superactions of the extract of Phyllostchys pubescens leaves, J. Food Biochem. 37 (2013) 501–509. [6] X. Li, X. Quyang, R. Cai, D. Chen, 3′,8″-Dimerization enhances the antioxidant capacity of flavonoids: evidence from acacetin and isoginkgetin, Molecules 24 (2019) 2039, [7] M.S.M. Al-Saleem, L.H. Al-Wahaib, W.M. Abdel-Mageed, Y.G. Gouda, H.M. Sayed, Antioxidant flavonoids from Alhagi maurorum with hepatoprotective effect, Phcog. Mag. 15 (2019) 592–599.


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