Accepted Manuscript Nicotine alkaloids as antioxidant and potential protective agents against in vitro oxidative haemolysis Karolina Malczewska-Jaskóła, Beata Jasiewicz, Lucyna Mrówczyńska PII:

S0009-2797(15)30132-0

DOI:

10.1016/j.cbi.2015.11.030

Reference:

CBI 7538

To appear in:

Chemico-Biological Interactions

Received Date: 2 July 2015 Revised Date:

28 October 2015

Accepted Date: 26 November 2015

Please cite this article as: K. Malczewska-Jaskóła, B. Jasiewicz, L. Mrówczyńska, Nicotine alkaloids as antioxidant and potential protective agents against in vitro oxidative haemolysis, Chemico-Biological Interactions (2015), doi: 10.1016/j.cbi.2015.11.030. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Nicotine alkaloids as antioxidant and potential protective agents against in vitro oxidative haemolysis

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Karolina Malczewska-Jaskółaa, Beata Jasiewicza*, Lucyna Mrówczyńskab*

Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznań, Poland

Department Cell Biology, Faculty of Biology, Adam Mickiewicz University, Umultowska

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89, 61-614 Poznań, Poland

Abstract

The capacity of eleven nicotine alkaloids to reduce oxidative stress was investigated. In order to provide a structure-activity relationships analysis, new nicotine derivatives with a

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substituent introduced into the pyrrolidine ring were synthesized and investigated together with nicotine and its known analogs. All newly synthesized compounds were characterized by 1

H, 13C NMR and EI-MS technique. The antioxidant properties of nicotine, its known analogs

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and newly produced derivatives, were evaluated by various antioxidant assays such 1,1diphenyl-2-picryl-hydrazyl free radical (DPPH•) scavenging, ferrous ions (Fe2+) chelating

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activity and total reducing ability determination by Fe3+→Fe2+ transformation assay. The protective effects of all compounds tested against 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) and tert-butyl hydroperoxide (t-BuOOH)-induced oxidative haemolysis and morphological injury of human erythrocytes, were estimated in vitro. The results showed that nicotine alkaloids exhibited various antiradical efficacy and antioxidant activity in a structure- and a dose-dependent manner. In addition, the capacity of nicotine

* Corresponding authors. Tel.: +48-61-8291695; fax.: +48-61-8291555. E-mail: [email protected] (Beata Jasiewicz), [email protected] (Lucyna Mrówczyńska)

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ACCEPTED MANUSCRIPT alkaloids to protect erythrocytes from AAPH- and t-BuOOH-induced oxidative haemolysis, was dependent on it`s incubation time with cells. Our findings showed that chemical and biological investigations conducted simultaneously can provide comprehensive knowledge concerning the antioxidant potential of nicotine alkaloids. This knowledge can be helpful in

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better understanding the properties of nicotine alkaloids under oxidative stress conditions.

Keywords: nicotine derivatives; free radicals; antioxidant properties; human erythrocytes;

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haemolysis; oxidative damage

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1. Introduction

Plant alkaloids represent one of the largest groups of natural products. A well-studied class of biologically active compounds includes (S)-(-)-nicotine (compound 1), the major alkaloid present in Nicotiana tabacum. It is a major pharmacologically active substance in tobacco and

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is the main cause of physiological addiction in smoking. Nicotine together with anabasine and anabasamine constitutes a group of natural ligands of nicotinic acetylocholine receptors (nAChRs). Anabasine (also called neonicotine) is a minor tobacco alkaloid established to be a

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selective α7-nAChRs agonist in an animal model with low toxicity for the potential treatment of schizophrenia [1-3]. It blocks the function of nAChRs via desensitization with a

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mechanism similar to that of nicotine. Anabasamine was shown to inhibit the catalytic activity of the enzyme acetylcholinesterase [4] and exhibit anti-inflammatory activity [5]. The action of nicotine has been extensively investigated in humans, and animals as well as in a variety of cell systems. It plays an important role in the developments of lung cancer, and cardiovascular disease in smokers [6-8]. Smoking is also considered to induce oxidative stress that can result in the oxidation of lipids, inactivation of certain proteins, disruption of biological membranes and induction of DNA single-strand breakage [9-10].

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ACCEPTED MANUSCRIPT Although many studies have investigated the antioxidant properties of nicotine the results still remain controversial. Several in vivo experiments suggest that the beneficial/protective effects of nicotine in both Parkinson's disease and Alzheimer's disease may be due to antioxidant mechanisms, while other studies have reported that nicotine induces oxidative

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stress in different tissues [11-13]. Recent studies showed that nicotine and cotinine analogs can be potential neuroprotective agents for Alzheimer disease [14].

In continuation of our interest in the chemistry and biology of natural-product-based

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compounds [15,16], a series of pyrrolidine-modified nicotine analogs was synthesized to elucidate the structure–antioxidant activity relationship. We applied three different

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antioxidant assays such 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH•) scavenging, ferrous ions (Fe2+) chelating activity and a total reducing ability determination by Fe3+→Fe2+ transformation assay to evaluate the antioxidant properties of newly synthesized compounds, as well as those of nicotine and its known analogs (compounds 1-11, see Fig. 1). Ferulic acid

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(FA), butylated hydroxytoluene (BHT) and Trolox were used as the reference antioxidants.

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Figure 1 about here

Additionally, the capacity of nicotine alkaloids to protect isolated human erythrocytes form

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oxidative damage induced by two free radicals inductors, namely hydrophilic 2,2'-azobis(2methylpropionamidine) dihydrochloride (AAPH) and hydrophobic tert-butyl hydroperoxide (t-BuOOH), was evaluated. Erythrocytes are the most abundant blood cells and are susceptible to oxidative damage because of their natural role as oxygen transporters. Under oxidative stress conditions the erythrocytes membrane cytoskeleton is damaged and as a consequence, specific cell shape transformations as well as haemolysis are observed [17-18]. The dual effects of nicotine in relation to oxidative damage and antioxidant as well as cytoprotection, including human erythrocytes, was reported [7,19], therefore performing a

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ACCEPTED MANUSCRIPT study with a series of nicotine alkaloids can shed some light on the relationship between the chemical structure and antioxidant properties of each compound. In our previous paper we showed that nicotine noticeably decreases the cell membraneperturbing potential of hydrophobic bile acids [15]. In this work we clarify the antioxidant

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potential of nicotine, its analogs and new derivatives using various antioxidant assays, including the cellular system. We aimed to evaluate the relationship between the chemical structure of each nicotine alkaloid and its effect on oxidative stress in vitro. This study

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represents the first estimation of eleven nicotine alkaloids, including those which are newly

2. Materials and methods

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synthesized, as antioxidant and human erythrocyte protective agents in vitro.

2.1. Chemicals and instruments

Starting materials and reagents used in reactions were obtained commercially from Aldrich

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and were used without purification. Proton nuclear magnetic resonance (1H-NMR) and Carbon-13 nuclear magnetic resonance (13C-NMR) spectra were recorded on a Varian 300/400MHz spectrometer. Chemical shifts are reported as δ values in parts per million (ppm)

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relative to tetramethylsilane (TMS) for all recorded NMR spectra. EI-MS mass spectra were recorded on 320MS/450GC Bruker mass spectrometer. The absorbance (Abs) was measured

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at the appropriate wavelength by using spectrophotometer SEMCO, EPOLL 2000 ECO (methods 2.3-2.8).

2.2. Synthesis and characterization of nicotine derivatives and analogs Synthesis of cotinine (2): Nicotine (162 mg, 1 mmol), mercury(II) acetate (478 mg, 1.5 mmol) and EDTA (438 mg, 1.5 mmol) were dissolved in 20 mL of water and refluxed for 18 hours. Brown solution was separate from metallic mercury and refluxed with 30% H3PO3 (20

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ACCEPTED MANUSCRIPT mL) for 2 hours. Potassium hydroxide was added to pH=7 and solution was extracted by CH2Cl2 until absence of alkaloids in organic phase (Dragendorff test). Combined organic phases was dried over MgSO4 and solution was evaporated. Brown oil was obtained. Yield: 87.4%. Anal. Calcd. for C10H12N2O:C, 68.18; H, 6.82; N, 15.91. Found: C, 68.20; H, 6.91; N,

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15.78. 1H NMR (300 MHz, CDCl3, TMS, ppm): δ 8.56 (1H-6’, ArH), 8.51 (1H-2’, ArH), 7.76 (1H-4’, ArH), 7.51 (1H-5’, ArH), 4.68 (1H-5), 3.40 (N-CH3), 2.45 (1H-4), 2.35 (1H-3), 2.31 (1H-3), 1.77 (1H-4).

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C NMR (75 MHz, CDCl3, TMS, ppm): δ 174.30, 149.12, 148.34,

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136.94, 134.70, 123.99, 60.87, 29.52, 27.50, 27.50.

Cotinine thio- (3) and seleno- (4) analogs were obtained according to the literature [20-21].

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Anabasamine (6) was obtained from anabasamine trichloride - Anabasamine x 3 HCl was dissolved in 2N HCl (5 ml). The solution was alkalized with KOH and (after cooling) extracted with diethyl ether. The ether solution was dried with KOH pellets, evaporated under pressure and oily residue was crystallized from MeOH. White crystalline 6 was obtained

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(96%), m.p. 64–65 °C. Anal. Calcd. for C16H19N3:C, 75.88; H, 7.51; N, 16.60. Found: C, 75.80; H, 7.48; N, 16.70. 1H NMR (300 MHz, CD3OD, TMS, ppm): δ 9.16 (1H-2’’, ArH), 8.63 (1H-2’, ArH), 8.59 (1H-6’’, ArH), 8.43 (1H-4’’, ArH), 7.94 (1H-4’, ArH), 7.92 (1H-5’,

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ArH), 7.57 (1H-5’’, ArH), 3.07 (1H-2), 3.02 (1H-6), 2.23 (1H-2), 2.05 (N-CH3), 1.84 (1H-4), 1.77 (1H-5), 1.76 (2H-3), 1.74 (1H-4), 1.66 (1H-5).

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S-benzylation reaction, general procedure: nicotine thiolactam (96 mg, 0.5 mmol), benzyl alcohol (162 mg, 1.5 mmol) or 2-/4-methoxy benzyl alcohol (207 mg, 1.5 mmol) were dissolved in dichloromethane and BF3 etherate (246 mg, 2 mmol) was added dropwise to the solution. The reaction mixture was stirred at room temperature for 72 h. The excess of dichloromethane was evaporated under reduced pressure and the residue was crystallized from acetonitrile to give 7-9 as a light yellow oils. Yields: 75, 70 and 82% for 7, 8 and 9, respectively.

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ACCEPTED MANUSCRIPT Compound 7: 1H NMR (300 MHz, DMSO-d6, TMS, ppm): δ 8.92 (1H-6’, ArH), 8.90 (1H-2’, ArH), 8.53 (1H-4’, ArH), 8.12 (1H-5’, ArH), 7.58-7.65 (2H-9,11, ArH), 6.98-7.33 (3H8,12,10, ArH), 4.87 (1H-5), 3.79 (2H-6), 3.73 (1H-3), 2.56 (N-CH3), 2.40 (1H-4), 1.84 (1H4).

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C NMR (75 MHz, DMSO-d6, TMS, ppm): δ 174.63, 161.29, 144.35, 141.57, 140.80,

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132.47, 132.32, 128.42, 128.16, 127.55, 126.67, 113.69, 113.49, 60.18, 29.26, 27.80, 27.03. EI-MS (m/z): 282 (C17H18N2S).

Compound 8: 1H NMR (300 MHz, DMSO-d6, TMS, ppm): δ 8.91 (1H-6’, ArH), 8.89 (1H-2’,

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ArH), 8.52 (1H-4’, ArH), 8.10 (1H-5’, ArH), 7.62 (2H-10,12, ArH), 7.01 (2H-9,11, ArH), 4.87 (1H-5), 3.79 (O-CH3), 3.03 (1H-3), 2.99 (2H-6), 2.57 (N-CH3), 2.36 (1H-4), 1.84 (1H-4). 13

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C NMR (75 MHz, DMSO-d6, TMS, ppm): δ 174.62, 156.72, 154.93, 141.12, 141.78, 140.98

132.33, 130.32, 129.71, 127.30, 120.18, 113.57, 110.54, 60.19, 55.10, 29.27, 27.86, 27.04. EIMS (m/z): 312 (C18H20N2OS), 192 (C10H12N2S).

Compound 9: 1H NMR (300 MHz, DMSO-d6, TMS, ppm): 8.88 (1H-6’, ArH), 8.84 (1H-2’,

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ArH), 8.46 (1H-4’, ArH), 8.28 (1H-5’, ArH), 7.44 (2H-8,12, ArH), 7.01(2H-9,11, ArH), 4.86 (1H-5), 3.78 (O-CH3), 3.08 (1H-3), 2.91 (2H-6), 2.56 (N-CH3), 2.37 (1H-4), 1.83 (1H-4). 13C NMR (75 MHz, DMSO-d6, TMS, ppm): δ 174.64, 159.36, 144.06, 141.83, 141.03, 130.82,

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129.31, 127.45, 126.66, 126.53, 125.28, 114.68, 113.91, 61.54, 55.26, 29.27, 27.86, 27.04. ESI-MS (m/z): 312 (C18H20N2OS), 192 (C10H12N2S).

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Titanium-mediated reductive alkylation of nicotine thiolactam, general procedure: Titanium(IV)iso-propoxide (426.3 mg, 1.5 mmol) was added to a stirred solution of the starting thiolactam 3 (192 mg, 1 mmol) in THF (20 mL) at 0 °C. A solution of the cyclopentylmagnesium chloride (515.5mg, 4 mmol) or cyclohexylmagnesium chloride (571.6 mg, 4 mmol) was then introduced dropwise, over 5 to 10 min at 0 °C. During the addition, the solution generally turned yellow, orange, brown and finally black. The cold bath was removed and the reaction mixture was stirred at 20 °C for 24 hours. H2O (1 mL) was then added and

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ACCEPTED MANUSCRIPT the flask was exposed to air, stirred until near complete decolouration. The mixture was filtered through a short pad with a layer of sand at the bottom, a layer of Na2SO4, and a layer of celite at the top, that was then rinsed with THF. The

resulting clear solution was

concentrated to afford the crude products. Yields: 60 and 70% for 10 and 11, respectively.

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Compound 10: 1H NMR (300 MHz, CDCl3, TMS, ppm): δ 8.50 (1H-6’, ArH), 8.45 (1H-2’, ArH), 7.71 (1H-4’, ArH), 7.34 (1H-5’, ArH), 3.97 (1H-5), 2.08 (N-CH3), 2.03 (1H-2), 1.871.14 (13H-3,4,6,7,8,9,10). 13C NMR (75 MHz, CDCl3, TMS, ppm): δ 148.83, 148.28, 139.40,

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134.55, 123.41, 68.92, 65.36, 40.86, 33.90, 29.81, 26.83, 25.82, 25.27, 25.12, 24.19. EI-MS (m/z): 230 (C15H22N2), 162 (C10H14N2).

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Compound 11: 1H NMR (300 MHz, CDCl3, TMS, ppm): δ 8.51 (1H-6’, ArH), 8.45 (1H-2’, ArH), 7.72 (1H-4’, ArH), 7.35 (1H-5’, ArH), 3.77 (1H-5), 2.07 (N-CH3), 2.01 (1H-2), 1.401.88 (4H-3,4), 1.88-0.92 (11H-6,7,8,9,10,11).

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C NMR (75 MHz, CDCl3, TMS, ppm): δ

148.83, 148.34, 134.59, 130.54, 123.64, 70.13, 68.88, 35.31, 30.36, 26.56, 26.38, 25.91,

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25.35, 24.52, 23.80, 23.78. EI-MS (m/z): 244 (C16H24N2), 192 (C10H12N2S), 162 (C10H14N2).

2.3. Fe3+ reducing power assay

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Reducing power was determined by the direct reduction of Fe3+(CN-)6 to Fe2+(CN-)6, and determined by measuring absorbance resulted from the formation of the Perl’s Prussian Blue

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complex following the addition of excess ferric ions (Fe3+). Different concentrations of compounds tested (0.01-1 mg/mL) in 0.06 mL of distilled water were gently mixed with 0.1 mL of 0.20 MPBS (pH 6.6) and 0.1 mL of 1% potassium ferricyanide [K3Fe(CN)6]. FA, Trolox and BHT were used as the reference compounds. The samples were vortexed and incubated for 20 min at 50°C. Following incubation, 0.1 mL of 10% trichloroacetic acid was added to the samples to acidify the reaction medium. Finally, 0.040 mL 0.6 M FeCl3 was added to the medium and the absorbance (Abs) was measured at 700 nm in a

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ACCEPTED MANUSCRIPT spectrophotometer. The increase in absorbance value of the reaction medium corresponded to more effective reduction capability of the compound tested. Each sample was made in

2.4. DPPH• free radical scavenging activity

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triplicate and three independent experiments were performed.

1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH•) shows absorbance at 517 nm which decreases upon reduction by an antioxidant. In brief, 0.1 mM solution of DPPH• was prepared

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in ethyl alcohol and 0.2 mL of this solution was added to 0.2 mL of compound tested at different concentrations (0.01-1 mg/mL) in ethyl alcohol and vortexed. FA, Trolox and BHT

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were used as the reference compounds. The samples were incubated in dark for 30 min at room temperature. Following incubation, the absorbance (Abs) was measured at 517 nm in a spectrophotometer. The percent DPPH• scavenging effect was calculated using the equation:

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DPPH• scavenging activity (%) = [(Abs0 – Absl)/Abs0] x 100 where Abs0 is the absorbance of the control reaction without compounds tested and Abs1 is

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the absorbance in the presence of compounds tested. Each sample was made in triplicate and

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three independent experiments were performed.

2.5. Ferrous ions (Fe2+) chelating activity Ferrous ions (Fe2+) chelating activity was evaluated by inhibition of the formation of Fe2+ferrozine complex after incubation of the compounds tested with Fe2+. Fe2+-chelating ability of compounds tested was determined by the absorbance of the ferrous ion-ferrozine complex at 562 nm. In brief, different concentrations of the compounds tested (0.01-1 mg/mL) in 0.2 mL ethyl alcohol were added to a solution of 0.6 mM FeCl2 (0.05 mL). EDTA was used as the

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ACCEPTED MANUSCRIPT standard metal chelator [22-24]. The reaction was started by the addition of 5 mM ferrozine (0.05 mL) in ethyl alcohol and shaken vigorously immediately. The samples were stored for 10 min at room temperature. Following incubation, the absorbance (Abs) of the solutions was

complex formation was calculated using the equation: Fe2+ chelating effect (%) = [1-(Abs1/Abs0)] x 100

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measured at 562 nm in a spectrophotometer. The percentage of inhibition of ferrozine–Fe2+

where Abs0 is the absorbance of the sample without the tested compound and Abs1 is the

2.6. Erythrocyte preparation

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independent experiments were performed.

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absorbance in the presence of compound tested. Each sample was made in triplicate and three

Freshly human erythrocytes suspensions were obtained from the blood bank. The erythrocytes were washed three times (3000 rpm, 10 min, +4°C) in 7.4 pH phosphate buffered saline (PBS

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– 137 mM NaCl, 2.7 mM KCl, 10 mM NaHPO4, 1.76 mM KH2PO4) supplemented with 10 mM glucose. After washing, cells were suspended in the buffer at 1.65x109 cells/mL, stored at

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+4°C and used within 5 h.

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2.7. Haemolysis assay under the compounds tested Erythrocytes (1.65x108 cells/mL, ~1.5% haematocrit) were incubated in PBS (7.4 pH) supplemented with 10 mM glucose and containing compounds tested in different concentrations (0.01, 0.1, 0.5, 1 mg/mL) for 60 min at 37°C in a shaking water bath. Samples with erythrocytes incubated in PBS without compounds tested were taken as the controls. Each sample was repeated three times and the experiments were repeated 4 times with erythrocytes from different donors. After incubation, the erythrocyte suspensions were centrifuged (3000 rpm, 10 min) and the degree of haemolysis was estimated by measuring the

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ACCEPTED MANUSCRIPT absorbance of the supernatant at 540 nm in a spectrophotometer. The results were expressed as percentage (%) of haemolysis. Haemolysis 0% was taken as the absorbance of the supernatant of erythrocyte suspensions in PBS only, while the total haemolysis (100%) was

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determined when PBS was replaced by distilled water. 2.8. Inhibition of induced-oxidative haemolysis

Erythrocytes (1.65x108 cells/mL, ~1.5% haematocrit) were incubated in PBS (pH 7.4)

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supplemented with 10 mM glucose and containing compounds tested in the concentration 0.01, 0.1, and 1 mg/mL for 20 min or 120 min at 37°C in a shaking water bath. After pre-

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incubation, 60 mM µM 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) or 250 µM tert-butyl hydroperoxide (t-BuOOH) were added, at final concentration 60 mM or 250 µM, respectively. Samples were incubated for next 4 h at 37°C in a shaking water bath. Erythrocytes incubated in PBS only and in the presence of AAPH or t-BuOOH, were taken as

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the controls. After incubation, the erythrocyte suspensions were centrifuged (3000 rpm, 10 min) and the degree of haemolysis was determined by measuring the absorbance (Abs) of the supernatant at 540 nm in a spectrophotometer. The percentage of inhibition was calculated

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using the following equation:

Inhibition of erythrocytes haemolysis (%) =

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100 – [ (Abssample – Absblank/Abscontrol – Absblank) x 100]

where Abssample is the absorbance value of supernatant obtained from samples incubated with compounds tested, Absblank is the absorbance of supernatant obtained from samples without compounds tested and AAPH or t-BuOOH, and Abscontrol is the absorbance of supernatant obtained from samples with AAPH or t-BuOOH and in the absence of compound tested. Each sample was made in triplicate and the results are presented as a mean value (±SD) of three independent experiments with erythrocytes from different donors.

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ACCEPTED MANUSCRIPT 2.9. Scanning electron microscope studies of erythrocyte shape Erythrocytes (1.65x108 cells/mL, ~1.5% haematocrit) were incubated in PBS (pH 7.4) supplemented with 10 mM glucose and containing compounds tested in the concentration 0.01, 0.1, and 1 mg/mL for 20 min or 120 min at 37°C in a shaking water bath. After pre-

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incubation, 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) or tert-butyl hydroperoxide (t-BuOOH) were added at final concentration 60 mM or 250 µM, respectively. Samples were incubated for next 4 h at 37°C in a shaking water bath. Erythrocytes incubated

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in PBS only and in the presence of AAPH or t-BuOOH, were taken as the controls. After incubation, cells were fixed in 0.1 glutaraldehyde for 1 hour at room temperature. Fixed cells

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were washed by exchanging of supernatant. The samples were gently vortexed and cell were fixed with 2% glutaraldehyde for 1 hour. After washed as above, cells were post-fixed with 1% OsO4 for 30 min at room temperature. The supernatant was exchange with PBS and samples were gently vortexed. Fixed cells were dehydrated in a series of ethanol solution

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(50%, 60%, 70%, 80%, 90%, 95%, and 100%), gold-sputtered, and examined using a EVO 40 (ZEISS, Germany) scanning electron microscope.

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2.10. Statistical analysis

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Data were analyzed statistically by “Analysis of Variance” (ANOVA) and groups were compared by Tukey Honsest Significant Differences (THSD) Test. p values lower than 0.05 were considered as statistically significant. To find significant differences between particular samples we created a boxplots for every sample ordered by different experimental conditions and run THSD to check whether those samples significantly differs at the level of confidence p FA = 7 > 8 > 9 = BHT > 3 > 10 > 4 > 11 = 5 > 6 >

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1 > 2.

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Figure 3 about here

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3.4. Chelating activity on ferrous ions (Fe2+) In this method, the Fe2+ form complex with ferrozine which is disturbed in the presence of chelating agents as standard metal chelator EDTA. As shown in Fig. 4, among all compounds used, nicotine (1), anabasine (5) and anabasamine (6) exhibited significant effective Fe2+chelating activity statistically similar to standard EDTA. Chelating activity of the most effective compounds as well as EDTA augmented with the increase in their concentration from 0.01 to 1 mg/mL.

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3.5. Protective activity on oxidative stress-induced haemolysis

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Initially, the effects of all compounds on RBC morphology and their membrane properties, namely their haemolytic activity, were tested. Neither nicotine nor it`s derivatives, induced significant modification in RBC shape or in cell membrane permeability up to the

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concentration 1 mg/mL after 60 min or 24 h incubation. Cells were mostly discocytic in shape and haemolysis up to 3-5% was obtained, similar to the control RBC (incubated with PBS

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only).

Fig. 5A summarize the inhibitory effect of nicotine alkaloids (20 min pre-incubated with RBC) on haemolysis induced by AAPH-derived radicals. Trolox as a positive control showed the highest protective activity, followed by the most effective compounds 2, 6 and 8-11. As

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seen from Fig. 5A, all compounds inhibited the AAPH-induced haemolysis in a significantly dose-dependent manner. Prolonging RBC pre-incubation with compounds from 20 min to 120 min (Fig. 5B), resulted in a noticeable increase of the inhibitory potency of compounds 3

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and 4 (more than a 50% inhibition of AAPH-induced haemolysis at concentration 1 mg/mL) and a decrease in the protective activity for all others. The reference Trolox was an exception.

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Interestingly, the reference BHT did not exhibit any protective effect after 120 min preincubation with RBC.

Figure 5 about here

The ability of all compounds preincubated with RBC for 20 or 120 min to inhibit tBuOOH-induced haemolysis were also analyzed (Fig. 6). After 20 min of pre-incubation, nicotine alkaloids did not protect RBC efficiently, with the exception of effective compounds

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ACCEPTED MANUSCRIPT 1 and 2 (Fig. 6A). Moreover, compounds 3, 4 and 5, as well as the reference BHT, did not inhibit t-BuOOH-induced haemolysis at all. The statistically significant difference between the most effective compounds 1 and 2 and the reference Trolox, was reported. As shown in Fig. 6B, the prolongation of RBC pre-incubation with compounds from 20 min to 120-min,

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did not result in an increase in their inhibitory potency against t-BuOOH-induced haemolysis, with the exception of compounds 3 and 4. Similar to results obtained after 20 min preincubation with RBC (Fig. 6A), compound 5 did not exhibit any protective effect after 120

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min of pre-incubation (Fig. 6B). Standard BHT was not effective as an antioxidant agent in

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the cellular system.

Figure 6 about here

stress conditions

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3.6. Protective activity against erythrocytes morphology alternations under oxidative

Normal discoid RBC (Fig. 7A) exposure to 60 mM AAPH (Fig. 7D-E) or 250 µM t-BuOOH

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(Fig. 7F) resulted in a echinocytic alteration of their morphology. These findings are in

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agreement with data presented in the field [26]. Moreover, egzovesicles released from echinocytic (spiculated) RBC was observed (Fig. 7 D-F). Numerous pores in the membrane of both sphere-shaped (spherocytic) (Fig. 7D) and discoid (Fig. 7E) RBC were detected. Changes in the erythrocytes shape induced by AAPH and t-BuOOH were prevented by all compounds tested in a structure- and dose-dependent manner. At the concentration 1 mg/mL, cotinine (compound 2) was able to significantly protect RBC against the echinocytic and spherocytic shape alteration induced by AAPH and t-BuOOH (compare Fig. 7 B-C with Fig.

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ACCEPTED MANUSCRIPT 7 D and F). These data are in accordance with the results obtained in the oxidative haemolysis studies (Fig. 5 and Fig. 6).

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Figure 7 about here

4. Discussion

The data concerning the antioxidant properties of nicotine are contradictory, as is it`s

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cytotoxic and genotoxic activity. It has been shown that the cellular effect of nicotine is strictly dose-dependent, namely high doses of nicotine are cytotoxic [13], including

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neurotoxicity [7]. Both cytotoxicity and genotoxicity of nicotine is mediated by an oxidative stress mechanism, dependent on its concentration and the exposition time of cells [27]. Conversely, low doses of nicotine, cotinine and their analogs which are characterized, at last in part, by the reduction of oxidative stress [11, 28] are widely studied to be used as

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antioxidant therapeutic agents in Parkinson's and Alzheimer's diseases. One reason for the used of nicotine in neurodegenerative diseases hinges upon its radical scavenging properties [29] and ferrous ions (Fe2+) chelating activity [28]; however, the neuroprotective activity of

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nicotine,cotinine and a series of it's analogs was also explained by direct action against amyloid-β-neurotoxicity [14]. On the other hand, the protective activity of nicotine against

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neurotoxicity induced by colchicine was caused by the effect of crosstalk between PI3K Akt and p38 or JNK signaling pathways [30]. The activity of antioxidants has been attributed to various mechanisms such as

prevention of chain initiation, binding of transition-metal ion catalysis, decomposition of peroxides, prevention of continued hydrogen abstraction, and reductive capacity and radical scavenging [31,32]. In order to obtain more insight into the antioxidant potential of a series of nicotine alkaloids, we applied various antioxidant assays, namely a Fe3+ reducing power assay, a DPPH• radical scavenging assay and a ferrous ions (Fe2+) chelation power assay, 16

ACCEPTED MANUSCRIPT including cellular system. The results of our research showed that nicotine and its derivatives exhibit antioxidant potential and cytoprotective activity which are significantly dependent on their structure and concentration. The reducing capacities of compounds are assessed by the extent of their conversion of

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a Fe3+/ferricyanide complex to a Fe+2/ferrous form which may serve as a indicator of its antioxidant power [33]. Namely, compounds with a reducing capacity may act as electron donors which could react with free radicals and effectively block radical chain reaction. As

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shown in Fig. 2, a significant improvement of the reducing capacity upon thionation or selenation of cotinine (compounds 3 and 4), similar to commercial antioxidants ferulic acid

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and Trolox, was observed in a dose-dependent manner. In addition, the reducing activity of selenoanalog was significantly higher than thioanalog in the range of concentration used (0.01-1 mg/mL). Our study on the modulation of antioxidant activity upon replacement of the carboxylic O atom of cotinine by its chalcolgen analogues S or Se shows that they are

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biologically interesting compounds.

Another assay, the DPPH• assay, has been widely used to evaluate the free radical scavenging effectiveness of various antioxidant substances. The effect of antioxidants on

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DPPH• radical scavenging was thought to be due to their hydrogen donating ability [34]. DPPH• is a stable free radical and accepts an electron or hydrogen radical to become a stable

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diamagnetic molecule [35]. The radical scavengers are free radical inhibitors and primary antioxidants. The degree of free radical scavenging activity of the investigated compounds is attributed to their structure. In the present study we claim that the high degree of radical scavenging activity of compounds 7-9 is due to the S-benzyl substitution and the presence of a double bond between the C2 and C3 carbon atoms in the pyrrolidine ring. As shown in Fig. 3, the DPPH• radical scavenging activity of compounds 7-9 significantly increased with increasing concentrations from 0.01 mg/mL up to 0.5 mg/mL.

17

ACCEPTED MANUSCRIPT Transition metal ions play an important role in the generation of oxygen free radicals in living organisms. Iron can exist in two oxidation states as ferric ion (Fe3+), which is a biologically inactive form, and as active ferrous ion (Fe2+), which possesses the ability to generate free radicals by losing or gaining of electrons. Depending on the conditions,

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particularly pH, ferric ion (Fe3+) can be reduced to active, pro-oxidant ferrous ion (Fe2+) which can be oxidized back through Fenton type reactions or Haber-Weiss reactions, with the production of hydroxyl radicals or superoxide anions respectively. The production of free

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radicals may lead to lipid peroxidation, protein modification, nucleic acids damage and finally to a number of diseases associated with oxidative stress. Therefore, chelating agents which

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chelate a metal ion can inhibit the metal-dependent formation of reactive oxygen species [36]. There is increasing evidence for the involvement of free radicals and iron (Fe2+)-induced oxidative stress in the pathogenesis of Parkinson's and Alzheimer's diseases [37]. It is believed that the capacity of nicotine to chelate Fe+2 prevents the production of •OH by the Fenton

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reaction. As can be seen from Fig. 4, all nicotine alkaloids possessed remarkable chelation power which generally increased as their concentration increased. Among the compounds investigated, anabasine and anabasamine (compounds 5 and 6) showed comparable levels of

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chelating activity to nicotine (compound 1), and were as effective as the standard metal chelator EDTA at all concentrations tested. The affinity of compounds 2-4 and 7-11 for

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ferrous ions was relatively low in comparison to EDTA. It seems that the presence of a substituent at the C2 position in the pyrrolidine ring makes the compounds less active than others in the series. Our earlier studies of nicotine and anabasine complexes with Zn(II) and Cu(II) ions in aqueous solution revealed that nicotine utilizes the pyridine nitrogen atom for this purpose [38]. In anabasine complexes the main coordination center is the piperidine nitrogen atom, while in zinc complexes also the pyridine nitrogen atom is also engaged to some small degree [39]. In summary, the iron (II) chelating activity of investigated

18

ACCEPTED MANUSCRIPT compounds is of great significance and can be considered for use in different chelation therapy, e.g. an iron overload in the body in cases of treatment of Thalassemia [40]. Based on the results above, different nicotine alkaloids were found to be effective antioxidants in various antioxidant assays when compared to reference antioxidants. Further

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research was performed to study both, the haemolytic activity and protective activity of all compounds using isolated red blood cells as a convenient cellular model. RBC are commonly used in the evaluation of the antioxidant properties of compounds because they are the most

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abundant blood cells and play a critical role in antioxidant protection in the blood [18-19,4145]. On the other hand, RBC cytoplasm is rich in oxygen and their membrane possesses a

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large amount of polyunsaturated fatty acids (PUFA), therefore, RBC can be a source of endogenous reactive oxygen species (ROS), such as peroxyl radicals (ROO•). In vivo exposure of RBC to ROS results in the peroxidation of the cell membrane components, and in consequence, lead to haemolysis [46] and the promotion of the oxidative stress in the

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cardiovascular system. It was confirmed that the RBC membrane of Alzheimer's disease patients is abnormally rich in peroxidized phospholipids [47]. Although RBC possess an endogenous antioxidant system consisting of three major enzymes (superoxide dismutase

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(SOD), glutathione peroxidase (GPx), and catalase (CAT), to protect them against ROS) [48], they use antioxidants obtained from the diet to increase their resistance to oxidative stress.

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Therefore, the synthesis of new natural-product-based compounds with antioxidant activity is the subject of many studies and is crucial for the further health-promoting applications [49]. The aim of the present study was the in vitro evaluation of the protective effect of nicotine alkaloids against oxidative damage in human RBC induced by free radicals derived from either ROO• generator as hydrophilic 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) or lipophilic tert-butyl hydroperoxide (t-BuOOH).

19

ACCEPTED MANUSCRIPT Haemolytic testing showed no membrane perturbing activity of nicotine alkaloids in the concentration rages used (up to 1 mg/mL), namely no accumulation of their molecules in the lipid bilayer of membrane. This finding is in agreement with data according to which nicotine can easily cross the cellular membrane barrier, without the incorporation into the exoplamic

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or cytosolic leaflet of the membrane [50]. Haemolysis induced by different compounds is a result of the incorporation of foreign molecules into the bilayer lipid structure and further formation of mixed micelles with cellular components [51]. Therefore, it could be concluded

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that all nicotine alkaloids used in this study did not alter the properties of RBC membrane up to concentrationj 1 mg/mL. However, it should be stressed that the cellular effect of nicotine

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is mainly related by nicotine acetylcholine receptors (nAChRs). Moreover, RBC are a simple cellular system lacking organelle, and the nicotine-induced cytotoxicity can be mediated for example by the endoplasmic reticulum stress pathway in nucleated cells [52]. As indicated by the results presented in Fig. 5, all nicotine alkaloids inhibited 60 mM

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AAPH-induced haemolysis in a structure-, dose-, and time-dependent manner. Compounds 2, 10 and 11 showed significant protection of erythrocyte against oxidative stress after 20 min pre-incubation time, whereas compounds 3 and 4 seem to be good inhibitors when their pre-

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incubation with RBC was prolonged to 120 min. Previous studies have shown [19] that AAPH-induced haemolysis was inhibited or increased by nicotine in a dose-dependent

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manner, respectively. Interestingly, in contrast to the results obtained in this study, cotinine increased haemolysis showing its prooxidant properties [19]. These differences can be explained by different experimental conditions and protocols, or, on the other hand, may clearly confirm the dual effect of nicotine on oxidative stress [7]. According to the literature, the hydrophilic radical initiator AAPH induces peroxidation of components in the outer layer of cell membrane, whereas lipophilic t-BuOOH promotes peroxidation of its inner layer [53]. As presented in Figures 5 and 6, RBC were protected by

20

ACCEPTED MANUSCRIPT nicotine alkaloids more efficiently from oxidative injury when ROO• were generated from AAPH and cells were attacked from outside. Therefore, the capacity of nicotine alkaloids to protect RBC from both oxidative-stress induced shape alteration (Fig. 7) and haemolysis (Fig. 5 and 6) can be explained by their direct action in the outer medium. The results obtained with

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RBC indicated that nicotine and its derivatives have the potential to protect the cell membrane against oxidative damage, however there is a weak correlation between their antioxidant activity proved in different antioxidant assays (section 3.1-3.4) and in a cellular system assay

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(section 3.5-3.6).

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5. Conclusion

The present study demonstrates that nicotine alkaloids exhibited antiradical and antioxidant properties in a structure- and dose-dependent manner, whereas their erythrocytes protective activity was also dependent on the incubation time with cells. In vitro non-cellular and

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cellular antioxidant assays showed significantly differ potential of the single compound studied, dependent on the method. Therefore, we conclude that chemical assays and cellular systems should be applied together to obtain a comprehensive evaluation of antioxidant

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properties of compounds tested. It can be stated that although nicotine alkaloids did not completely inhibit oxidative haemolysis in vitro, further structure-activity studies may help to

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obtain nicotine-based effective antioxidants.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

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ACCEPTED MANUSCRIPT This work was financially supported by the funds of Faculty of Chemistry, Adam Mickiewicz University, and by the statutory activity No. S/P-B/004 of the Department of Cell Biology, Faculty of Biology, Adam Mickiewicz University. The authors thank MSc. Eng. Aleksander

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Ratajczak for his excellent scanning electron microscopy assistance.

Supplementary data

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Supplementary data associated with this article can be found, in the online version.

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Figures legend Figure 1. Chemical structure of compounds investigated

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Figure 2. Fe3+– Fe2+ reductive potential of compounds tested, and standard antioxidants ferulic acid (FA), Trolox and BHT at different concentrations. The numbers are, respectively: 1-nicotine; 2-cotinine; 3-thio-analogs of cotinine; 4-seleno-analogs of cotinine; 5-anabasine; 7-2-benzylthionicotine;

methoxybenzylthio)-nicotine;

8-2-(2-metoxybenzylthio)-nicotine;

10-2-(cyclopentyl)-nicotine;

9-2-(4-

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6-anabasamine;

11-

2-(cyclohexyl)-nicotine.

Results are presented as average ± SD (n=3). Different letters indicate samples that were

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significantly different (p < 0.05)

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Figure 3. DPPH free radical scavenging activity of compounds tested and standard antioxidants ferulic acid (FA), Trolox and BHT at different concentrations. Results are presented as average ± SD (n=3). Different letters indicate samples that were significantly

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different (p < 0.05). Other indications as in Figure 2

Figure 4. Ferrous chelating activity of compounds tested, standards ferulic acid (FA), Trolox, BHT and EDTA as positive control at different concentrations. The results are average of the

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individual experiment. EDTA were used as as a positive control. Results are presented as average ± SD (n=3). Different letters indicate samples that were significantly different (p

Nicotine alkaloids as antioxidant and potential protective agents against in vitro oxidative haemolysis.

The capacity of eleven nicotine alkaloids to reduce oxidative stress was investigated. In order to provide a structure-activity relationships analysis...
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