Chemico-Biological Interactions 212 (2014) 40–46
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Novel oxime based flavanone, naringin-oxime: Synthesis, characterization and screening for antioxidant activity Mustafa Özyürek a,⇑, Damla Akpınar a, Mustafa Bener a, Baki Türkkan b, Kubilay Güçlü a, Resßat Apak a a b
Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar, Istanbul, Turkey Department of Chemistry, Faculty of Science and Letters, Harran University, Osmanbey, Sßanlıurfa, Turkey
a r t i c l e
i n f o
Article history: Received 31 July 2013 Received in revised form 3 December 2013 Accepted 29 January 2014 Available online 6 February 2014 Keywords: Naringin Naringin oxime Flavanone oximes Antioxidant activity Cupric reducing antioxidant capacity (CUPRAC) assay
a b s t r a c t Recent interest in polyphenolic antioxidants due to their involvement in health benefits has led to the investigation of new polyphenolic compounds with enhanced antioxidant activity. Naringin (40 ,5,7-trihydroxyflavanone-7-b-L-rhamnoglucoside-(1,2)-a-D-glucopyranoside) is one of the major flavanones in citrus and grapefruit. The present study aimed to synthesize naringin oxime from naringin and to evaluate its antioxidant and anticancer potential using in vitro assay system. The structure of the synthesized compound, naringin oxime, was elucidated by FT-IR, 1H NMR, elemental analysis and UV–vis spectroscopy. Antioxidant capacity of naringin oxime, as measured by the cupric reducing antioxidant capacity (CUPRAC) method, was found to be higher than that of the parent compound naringin. Other parameters of antioxidant activity (scavenging effects on OH, O 2 , and H2O2) of naringin and naringin oxime were also determined. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Flavonoids as an important group of natural substances have variable phenolic structures, and are found in abundance in fruits, vegetables, grains, flowers, wine, and tea [1]. They possess beneficial effects against serious diseases, such as cancer, cardiovascular disease, and neurodegenerative disorders [2]. In vitro experimental studies show that flavonoids act as antioxidants, antimicrobials, antivirals, and antiinflammatories [3]. Naringin (40 ,5,7-trihydroxyflavanone-7-b-L-rhamnoglucoside-(1,2)-a-D-glucopyranoside) is a member of the flavonoid family that shows various bioactivities on human health as antioxidant, reactive oxygen species (ROS) scavenger, antiinflammatory and antiapoptosis agent [4] having anti-carcinogenic [5] and neuro-protective effects [6], and especially a most investigated cancer preventive agent [7]. Naringin is found in abundance in citrus, grapefruit and juices [8]. When naringin is taken orally, it is metabolised by the enzymes a-rhamnosidase and b-glucosidase to its aglycone naringenine, which is in the more absorbable form [9,10], because naringin kinetically exhibits a delay in its intestinal absorption, resulting in decreased bioavailability. Naringin is a naturally available antioxidant that can be used for the synthesis of other novel antioxidants for ⇑ Corresponding author. Address: Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar, 34320 Istanbul, Turkey. Tel.: +90 212 473 7070x17627; fax: +90 212 473 7180. E-mail address: [email protected] (M. Özyürek). http://dx.doi.org/10.1016/j.cbi.2014.01.017 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
enhancing antioxidant activity. Pereira et al. synthesized a new naringin–metal complex, naringin–Cu(II), and this compound was found to possess higher antioxidant, antiinflammatory and tumor cell cytotoxicity activities than free naringin [11]. Oximes (Ox) (R1R2C@NOH) constitute an important class of hydroxylamines, where R1 is organic side chain and R2 is either hydrogen, forming an aldoxime, or another aromatic group, forming ketoxime [12]. Oximes and their derivatives are important intermediates in organic syntheses [13]. The oxime functional group can easily be bound to such important organic groups as carbonyl, amino, nitro and cyano functions and can also serve as a convenient protective group [14]. Oximes have been used for many important pharmaceutical and synthetic chemistry applications, and often act as chemical building blocks for the synthesis of agrochemicals and pharmaceuticals [12]. Oxime-type functional groups are included in many organic medicinal agents used in the treatment of organophosphate (OP) poisoning. Inhibition of acetylcholinesterase (AChE) results from acute OP toxicity, whereas oximes, by reactivating AChE, are considered to be rather effective against OP poisoning [15,16]. Therefore, synthesis and investigation of various oximes have an important role in medicinal research. Puntel et al. investigated the capacity of butane-2,3-dionethiosemicarbazone oxime to scavenge different forms of reactive species (RS) both in vivo and in vitro, and found significant hydrogen peroxide (H2O2), nitric oxide (NO) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity for the oxime [16]. The antiradical and
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antioxidant activities of four biologically active N,N-diethyloaminoethyl ethers of flavanone oximes were investigated by Metodiewa et al., and these compounds were shown to act as promising antioxidants and radioprotectors comparable to rutin activities under oxidative stress conditions [17]. Naringenin oxime was investigated for its antioxidant capacity by using the cupric reducing antioxidant capacity (CUPRAC) method, where the oxime functional group significantly enhanced the antioxidant capacity of pure naringenin [18]. Because of the important advantageous uses of oximes, especially of flavanone oximes, we synthesized naringin oxime (Fig. 1) and determined its antioxidant properties by using the CUPRAC method [19]. The chromogenic oxidizing reagent of the CUPRAC assay, bis(2,9-dimethyl-1,10-phenanthroline)copper(II) (abbreviated as Cu(II)-Nc), is simple, diversely applicable to both hydrophilic and lipophilic antioxidants, stable and easily available at low cost [19]. The CUPRAC method has been successfully applied to the determination of antioxidants in food plants (apricot, herbal teas, wild edible plants, herby cheese, etc.), natural dyes [20], and to human serum [21]. The main CUPRAC method was modified for measuring the hydroxyl radical scavenging activities of polyphenolics [22], xanthine oxidase (XO) inhibition activity [23], hydrogen peroxide scavenging activity of polyphenolics in the presence of Cu(II) catalyst [24], and development of a CUPRACbased antioxidant sensor on a Nafion membrane [25]. The synthesis reaction of naringin oxime compound is shown in Fig. 2. The antioxidant activities (scavenging effects on OH, O 2 , and H2O2) of naringin oxime have also been investigated. Scavenging activities against these reactive species were tested, because OH generated in the human body shows the strongest oxidative activity among ROS, H2O2 as a non-radical oxidizing species may be generated in tissues and diffuse across biological membranes, and O 2 has a great importance as a physiological signaling molecule while its scavengers can combat against many types of diseases [26,27]. The chelating ability toward transition metal ions is an important mechanism of secondary antioxidant action, because substitution of a C-4 (@O) group in the parent compound structure with a C-4 (@NAOH) group in the oxime derivative may enhance the antioxidant activity of the product through stronger binding of transition metal ions [18], thereby preventing Fenton-type reactions of reactive species generation. Further research is needed to show the possible pharmacological and biological activity of this newly synthesized naringin oxime compound, and this work is believed to potentiate the development of some new oxime-based antioxidants with enhanced antioxidant activity. 2. Materials and methods 2.1. Materials and apparatus All reagents and solvents were of analytical reagent grade. Naringin (40 ,5,7-trihydroxyflavanone-7-rhamnoglucoside) and
neocuproine (2,9-dimethyl-1,10-phenanthroline) were purchased from Sigma Chemical Co. (Steinheim, Germany). Copper(II) chloride dihydrate, ammonium acetate (NH4Ac), absolute ethanol (EtOH), methanol (MeOH), dimethyl sulfoxide (DMSO), hydroxylamine hydrochloride, sodium acetate trihydrate were purchased from Merck (Darmstadt, Germany). All chemicals used in reagent preparation were weighed with an accuracy of ±0.0001 g. The spectra and absorption measurements were recorded in matched Helma quartz cuvettes using a Varian CARY Bio 100 UV–vis spectrophotometer. The elemental analyses were performed using a CHNS instrument model Carlo-Erba 1106 elemental analyzer. 1H NMR spectra in DMSO were obtained on a Varian INOVA 500 MHz NMR spectrometer. The IR spectra were obtained by using KBr pellets in the spectral range 4000–400 cm1 on a Perkin Elmer RXI FT-IR spectrometer. Melting points were found with a Stuart Scientific SMP3 Melting Point Apparatus. 2.2. Preparation of solutions The standard solutions of naringin oxime and naringin were prepared in EtOH at a concentration of 1.0 mM. All standard solutions were stored at +4 °C prior to analysis. The CuCl2 solution (10.0 mM) and ammonium acetate buffer solution (1 M, pH 7.0) were prepared in distilled water and neocuproine solution (7.5 mM) in absolute ethanol. 2.3. Cell culture and treatment The canine mammary carcinoma cell line CMT-U27 (a generous gift from Assoc. Professor Eva Hellmén) was obtained from Uppsala University, Sweden. CMT-U27 cells were cultured in DMEM-F12 (Sigma Chemicals, St. Louis, USA), supplemented with 10% fetal bovine serum (Biological Industries, Israel), 100 IU mL1 penicillin G, 100 lg mL1 streptomycin, and 2.5 lg mL1 amphotericin B (Sigma, St. Louis, USA), at 37 °C in a humidified 5% CO2 atmosphere. Stock solution of naringin oxime was freshly prepared in DMSO and diluted with DMEM-F12 to a final concentration 60.1% of DMSO. Control groups received DMSO vehicle at a concentration equal to that in naringin oxime treated cells. 2.4. Synthesis of naringin oxime Naringin oxime was prepared by treating 0.01 mol (5.8 g) naringin with hydroxylamine hydrochloride (0.01 mol, 0.69 g) in 25 mL ethanol and sodium acetate trihydrate (0.01 mol, 1.36 g) in water. The solution was heated at reflux on a water bath for 4 h with constant stirring. On cooling to room temperature and adding 10 mL dichloromethane to solutions, a white solid precipitated out. It was filtered, washed with water, and dried over P2O5 under vacuum. The product yield was 60%. Melting point: 203 °C. Elemental OH
OH HO H3C
HO
OH O
O O 7
HO HO HO
O CH2
1
8
6
9 O 2 10
5 OH
Naringin
2'
O
4
3
1'
3'
'
6
4' 5'
OH
H3C
OH O
O O 7
HO O
HO HO
CH2
6
8
1 9 O 2
1'
10 4 3 5 OH N
Naringin oxime Fig. 1. The structures of naringin and naringin oxime.
2'
OH
3' 4' OH 6'
5'
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HO
HO
O
O
O
OH
NH 2OH. HCl /CH3COONa/MeOH
OH
4-5 h, rf
O
O
O HO
O
O
HO
OH HO
O HO HO
OH
N
OH
CH3
O OH
HO
O HO HO
OH
CH3
Fig. 2. Schematic representation of the synthesis of naringin oxime.
analysis results were; found (%): C 54.19; H 5.20; N 2.23; calculated for [C27H33NO14] [atomic mass 595.55]: C 54.45; H 5.59; N 2.35%. 2.5. Antioxidant capacity of naringin oxime measured by normal CUPRAC measurement The CUPRAC method is based on the reduction of light-blue colored cupric neocuproine complex (Cu(II)–Nc) by antioxidants to the yellow-orange colored cuprous chelate (Cu(I)–Nc) [19]. To a test tube were added 1.0 mL each of Cu(II), Nc, and NH4Ac buffer solutions. Antioxidant standard solutions (x) mL and H2O (1.1x) mL were added to the initial mixture so as to make the final volume (4.1 mL). The tubes were stoppered, and after 30 min standing at room temperature, the absorbance at 450 nm (A450) was recorded against a reagent blank. The standard calibration curves of each antioxidant compound was constructed in this manner as absorbance vs concentration, and the CUPRAC molar absorptivity of each antioxidant was found from the slope of the calibration line concerned. The scheme for normal measurement of antioxidants is summarized as: 1.0 mL Cu(II) + 1.0 mL Nc + 1.0 mL NH4Ac buffer + (x) mL antioxidant + (1.1x) mL H2O; total volume = 4.1 mL, measure A450 against a reagent blank after 30 min of reagent addition. 2.6. Incubated sample measurement by the CUPRAC method The mixture solutions containing sample and reagents were prepared as described in ‘normal measurement’; the tubes were stoppered and incubated for 20 min in a water bath at a temperature of 50 °C. The tubes were cooled to room temperature under running water, and their A450 values were measured [19]. 2.7. Hydroxyl radical scavenging (HRS) activity Hydroxyl radicals (OH) in aqueous media were generated through the Fenton system and spectrophotometrically determined via hydroxylation of a salicylate probe by the modified CUPRAC method [22]. To a test tube were added 1.5 mL of phosphate buffer (pH 7.0), 0.5 mL of 10 mM sodium salicylate, 0.25 mL of 20 mM Na2–EDTA, 0.25 mL of 20 mM FeCl2 solution, (2.0x) mL H2O, (x) mL naringin oxime solution (x varying between 0.1 and 0.5 mL) at a concentration of 2.0 105 M, and 0.5 mL of 10 mM H2O2 rapidly in this order. The mixture in a total volume of 5.0 mL was incubated for 10 min in a water bath kept at 37 °C. At the end of this period, the hydroxyl radical generation reaction was stopped with adding 0.5 mL of 268 U mL1 catalase solution,
and vortexed for 30 s. Final mixtures (0.5 mL of the incubation solution) were subjected to the HRS-CUPRAC method [22].The inhibition ratio of naringin oxime (%) was calculated using the equation:
Inhibition ratio ð%Þ ¼ 100
A0 A A0
ð2:1Þ
where A0 and A are the CUPRAC absorbances of the system in the absence and presence of scavenger, respectively. 2.8. Hydrogen peroxide scavenging (HPS) activity The ability of naringin oxime to scavenge hydrogen peroxide was determined according to the method of Özyürek et al. [24]. To a test tube were added 0.7 mL of phosphate buffer (pH 7.4), 0.4 mL of 1.0 mM H2O2, 0.4 mL of 0.1 mM CuCl22H2O in this order (H2O2 incubation solution, used as reference). To the other two test tubes were added 0.5 mL of phosphate buffer (pH 7.4), 0.4 mL of 1.0 mM H2O2, 0.2 mL naringin oxime solution, and 0.4 mL of 0.1 mM CuCl22H2O solution rapidly in this order (named as scavenger solutions-I and II; scavenger solutions-I and II, identical at this stage). The mixtures in a total volume of 1.5 mL were incubated for 30 min in a water bath kept at 37 °C. At the end of this period, to both reference and scavenger solution-I was added 0.4 mL H2O, and to scavenger solution-II was added 0.4 mL of 268 U mL1 catalase solution (the contents of scavenger solutionI and -II became different at this stage), and vortexed for 30 s. Final mixtures (1.0 mL of the incubation solution) were subjected to the HPS-CUPRAC method [24]. The HPS activity of naringin oxime (%) was calculated using the equation:
HPS ð%Þ ¼ 100
A0 ðA1 A2 Þ A0
ð2:2Þ
where A0 is the CUPRAC absorbance of reference H2O2 incubation solution, A1 and A2 are the CUPRAC absorbances of scavenger solutions-I and -II, respectively. 2.9. Superoxide anion radical scavenging activity The superoxide anion radicals (O 2 ) were generated in vitro in a non-enzymatic system (PMS-NADH) and determined spectrophotometrically by nitroblue tetrazolium (NBT) reduction method [28]. To a test tube were added (2.5x) mL DMSO, (x) mL (usually, x = 0.5) scavenger solution at a concentration of 1.0 103 M, 2.0 mL of 468 lM NADH, 1.0 mL of 300 lM NBT, in this order. The reaction was started by adding 1.0 mL of 60 lM PMS solution to the incubation mixture. The mixture in a total volume of 6.5 mL
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was incubated for 5 min in a water bath kept at 25 °C, and the absorbance was read at 560 nm against DMSO. Decreased absorbance of the incubation reaction mixture indicated increased superoxide anion radical scavenging activity. All experimental results were expressed as the mean ± standard deviation (S.D.) of triplicate determinations. The inhibition ratio of naringin oxime (%) was calculated using the equation:
Inhibition ratio ð%Þ ¼ 100
A0 A A0
ð2:3Þ
where A0 and A are the absorbances of the incubation reaction mixture in the absence and presence of scavenger, respectively. 2.10. Cell proliferation assay CMT-U27 cells were cultured in 96-well plates at a density of 1 104 cells/100 lL medium and allowed to attach for 24 h. Thereafter, medium was removed and replaced with 100 lL of medium containing various concentrations (0–1000 lM) of naringin oxime and incubated for 48 h. After incubation, cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) cell proliferation kit (Roche, Germany) [29]. Briefly, 10 lL of MTT solution 5 mg mL1 was added to each well and incubated for 4 h at 37 °C in CO2 incubator. The purple water insoluble formazan salt was then dissolved with 10% SDS in 0.01 M HCl and incubated overnight in a humidified 5% CO2 atmosphere. The optical densities (OD) of the wells were measured at 595 nm by microplate reader (Molecular Devices, USA). Cell proliferation inhibition rate (CPIR) was calculated using the following equation: CPIR, % = 100 [1 (average OD value of the treated wells)/(average OD value of the control wells)]. The effect of each compound on growth inhibition was assessed as percent cell viability where vehicle-treated cells were taken as 100% viable. 3. Results and discussion 3.1. Physical properties of naringin oxime The cream compound was stable at room temperature. It was soluble in MeOH, EtOH, and DMSO, but insoluble in water and DCM.
Fig. 3. UV–vis spectra of naringin and naringin oxime in ethanol.
3.3.1. Naringin 1 H-NMR (500 MHz, DMSO-d6): 12.32 (d, J = 3.25 Hz, 5-OH), 9.57 (s, 40 -OH), 7.31 (2H, m, H-20 and H-60 ), 6.79 (2H, dd, J = 1.49 and J = 1.39 Hz, H-30 and H-50 ), 6.10 (1H, brs, H-6), 5.49 (1H, m, H-2), 4.67 (1H, d, J = 4.64 Hz, H-3 ax), 4.60 (1H, d, J = 4.26 Hz, H-3 eq), 3.31 (1H, s, ACH2), 3.26 (1H, s, ACH2), 1.15 (3H, d, ACH3). 3.3.2. Naringin oxime 1 H-NMR (500 MHz, DMSO-d6): 12.03 (s, 5-OH), 11.34 (s, N-OH), 9.58 (s, 40 -OH), 7.32 (2H, m, H-20 and H-60 ), 6.79 (2H, d-d, J = 1.57 Hz, J = 1.34 Hz, H-30 and H-50 ), 6.10 (1H, t, H-6), 6.07 (1H, t, H-8), 5.49 (1H, m, H-2), 4.68 (1H, d, J = 4.57 Hz, H-3 ax), 4.61 (1H, d, J = 4.10 Hz, H-3 eq), 2.53 (1H, s, ACH2), 2.45 (1H, s, ACH2), 1.15 (3H, d, ACH3). The 1H NMR spectrum of naringin oxime exhibited very similar signals to that of naringin with very small chemical shift differences being in the interval 0.30–0.42 ppm for aromatic protons. There was an additional OH proton signal, observed at d 11.34, which was attributed to the oxime hydroxyl (C@NAOH). The presence of this hydroxyl was supported by the FT-IR spectrum (3395 cm1). 3.4. FT-IR study of naringin oxime
3.2. UV–vis spectroscopic study of naringin oxime The UV–vis spectra of naringin and naringin oxime in EtOH are shown in Fig 3. Naringin, like most flavones and flavonols, exhibits two major absorption bands in the UV region, at 284 nm (band I) representing B-ring absorption (cinnamoyl system), and at 229 nm (band II), considered to be associated with the absorption involving the A ring benzoyl system [30]. Compared to naringin absorption spectrum, band II of the naringin oxime compound is bathochromically shifted (i.e. to the longer wavelength region) as shown in Fig. 3, possibly due to added intra-molecular H-bonding alternatives between the C-4 (@NAOH) and C-5 (OH) substituents. Considering the greater hypsochromic shift of band I observed in the oxime derivative of naringenin relative to parent naringenin (i.e. from 294 nm of naringenin to 284 nm of naringenin oxime) [18], it can be deduced that the 7-b-L-rhamnoglucoside functionality of naringin reduced the spectral differences between naringin and naringin oxime.
The FT-IR spectra of naringin and naringin oxime are shown in Fig. 4. All the spectra are characterized by vibrational bands mainly due to the OH, CAH, C@O, C@N, and C@C functional groups. The t (OH), t (CAH), t (C@O), t (C@C) for naringin appear at 3425 cm1, 2957–2859 cm1, 1645 cm1, and 1597–1502 cm1, respectively. The t (OH), t (CAH), t (C@N), t (C@C) for naringin oxime appear at 3395 cm1, 2970–2875 cm1, 1620 cm1, and 1582–1520 cm1, respectively. The naringin band at 1645 cm1 may be assigned to t (C@O) stretching, whereas in naringin oxime, the band at 1620 cm1 assignable to t (C@N) confirms the presence of NAOH group bound to naringin. Türkkan et al., in reporting the synthesis and characterization of naringenin oxime, observed that the naringenin band at 1640 cm1 assigned to t (C@O) was shifted to 1628 cm1 assignable to t (C@N) when the oxime group was inserted to the main structure [18]. 3.5. Antioxidant capacity of naringin and naringin oxime measured by the CUPRAC method
3.3. 1H NMR study of naringin oxime The 1H NMR (DMSO) data of naringin and naringin oxime are separately given below:
The reaction equation for the CUPRAC method: Cu(II)–Nc with n-electron reductant phenolic antioxidants [19] can be formulated by:
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M. Özyürek et al. / Chemico-Biological Interactions 212 (2014) 40–46
Transmittance (%)
(a)
4000
(b)
3500
3000
2500
2000
1500
1000
500
Wave Number (cm ) -1
Fig. 4. The FT-IR spectra of solid compounds: (a) naringin oxime; (b) naringin.
þ þ nCuðNcÞ2þ 2 þ ArðOHÞn $ nCuðNcÞ2 þ Arð@OÞn þ nH
ð1Þ
The CUPRAC method [19] was applied to naringin and naringin oxime as two interrelated procedures, i.e., normal (at room temperature) and incubated (at 50 °C) versions of the assay, in comparison with the trolox standard reference compound. The linear calibration equations of naringin oxime and naringin (as absorbance in a 1-cm cell vs molar concentration) gave the molar absorption coefficient (e) as the slope (Table 1); the linear working ranges over which Beer’s law was valid are given in the same table. The CUPRAC molar absorption coefficient of the tested antioxidant divided by that of trolox under the same conditions gave the (unitless) trolox equivalent antioxidant capacity (TEAC), or TEAC coefficient of that antioxidant (Table 1). Fig. 5 shows the CUPRAC reaction kinetics with individual antioxidants measured at room temperature. It is apparent from Fig. 5 that naringin and naringin oxime showed a gradual absorbance increase with time, which determined the time period of measurement (i.e., 30 min after the mixing of reagent with the analytes). As the reduction potential of the antioxidant approaches that of the reagent, the thermodynamic efficiency and possibly the rate of the oxidation reaction decreases, which is the case for naringin (Ered o = 0.6 V). The anodic peak potential of the first oxidation wave of naringenin (i.e. the aglycon of naringin) was reported as 0.688 V [31]. In the CUPRAC method, the limit of quantification (LOQ) for naringin and naringin oxime were determined to be 22.7 lM and 0.75 lM, respectively, enabling a much higher sensitivity for the latter. The calibration curves of naringin and naringin oxime (CUPRAC absorbances vs antioxidant concentrations) are shown in Fig. 6. All of the easily oxidized flavonoids exhibited standard reduction potentials of 60.2 V, whereas naringin, having a potential þ close to that of the CuðNcÞ2þ 2 CuðNcÞ2 couple [19], underwent a slow reaction with the reagent. Naringin oxidation could only be enhanced after 50 °C incubation in the CUPRAC method, but its TEAC coefficient still remained low (i.e. increased from 0.02 to
Fig. 5. CUPRAC reaction kinetics with individual antioxidants; rate of increase in absorbance at 450 nm for 1 mM solutions of naringin and naringin oxime (at room temperature).
0.13 with incubation, Table 1). Regarding the antioxidant efficiency of flavanones, Yuting et al. tested several flavonoids in the inhibition of lipid peroxidation in mouse liver homogenates, and found several micromolar IC50 values for rutin, morin, and quercetin, but no inhibition for the flavanones including naringin, confirming that the antioxidant activity of naringin is not of the same magnitude as of common flavonoids [32]. On the other hand, the TEAC coefficients of naringin oxime measured with the normal and incubated CUPRAC methods (TEACN = 0.52 and TEACInc = 4.70, Table 1) were found to be much higher than the corresponding values of naringin, probably as a result of increased stabilization of the 1-e oxidized product (i.e., nitroxyl-aryloxyl radical) of naringin oxime as compared to that of naringin by intra-molecular H-bonding [33]. As the reduction potential of the redox pair: {1e oxidized product/parent compound} is lowered in flavonoids due to stabilization of the aryloxyl radical, the antioxidant power of the concerned compound (in this case, naringin oxime) is enhanced [34]. 3.6. Hydroxyl radical scavenging (HRS) activity Hydroxyl radical (OH), which can easily react with amino acids, DNA and membrane components, shows the strongest chemical reactivity among ROS. HRS of naringin oxime as a function of lM concentration with respect to Eq. (2.1) using the modified CUPRAC method (HRS-CUPRAC) [22] is given in Fig. 7. Naringin oxime showed statistically (at 95% confidence level) similar HRS, the effect being (79.9 ± 1.6)% at the initial concentration of 20 lM, compared to that of naringin (81.5 ± 0.7)% at the same concentration (N = 3), and HRS activities were dose-dependent. Seemingly, the replacement of 4-keto functionality in naringin with 4-NOH in naringin oxime does not make a difference in the HRS activities of the two compounds, as HRS action is inherently less structurespecific than other similar assays of ROS quenching due to the very high rates of hydroxyl radical reactions with biological substrates at the second-order rate constants of 108109 M1 s1.
Table 1 The molar absorption coefficients, linear ranges and TEAC coefficients of naringin and naringin oxime with respect to the CUPRAC method (normal (N) and incubated (Inc.) CUPRAC methods). Antioxidant
Naringin Naringin oxime
Molar absorption coefficient e (L mol1 cm1) CUPRACN
CUPRACInc.
3.3 102 8.6 103
2.2 103 7.8 104
Linear range (mol L1)
1.5 104 – 3.7 103 6.3 106 – 1.5 104
TEACCUPRAC TEACN
TEACInc
0.02 0.52
0.13 4.70
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Antioxidants are able to inhibit formazan formation [28]. The decrease of absorbance at 560 nm with antioxidants indicates the consumption of O 2 in the reaction mixture. The percentage inhibition of O 2 by 1 mM concentrations of naringin oxime and naringin oxime were found as (6.8 ± 0.6)% and (4.8 ± 0.5)%, respectively. 3.9. The effects of naringin oxime on cell proliferation Cell viability was assessed by MTT assay based on the conversion of the water soluble MTT to an insoluble colored formazan product by the active mitochondria of living cells [29]. After 48 h incubation, naringin oxime inhibited cell proliferation at 100, 200, 400 and 1000 lM by 10.39%, 11.79%, 12.7% and 17.85% of the control level, respectively. However, IC50 value for naringin oxime was not observed in the concentration range studied, and the corresponding IC50 value could not be calculated.
Fig. 6. The calibration curves as absorbance vs concentration of naringin oxime and naringin (inset) with respect to the normal CUPRAC method.
3.7. Hydrogen peroxide scavenging (HPS) activity The ability of naringin oxime to scavenge hydrogen peroxide was measured by the HPS-CUPRAC method [24] and compared to that of naringin. Hydrogen peroxide scavenging (HPS) activity of naringin oxime at 1 mM (initial conc.) was found to be (3.3 ± 0.1)%. On the other hand, naringin exhibited (26.0 ± 1.2)% HPS activity at the same concentration (N = 3). Since HPS-CUPRAC is a transition metal ion- (specifically Cu(II)-) catalyzed assay involving H2O2 degradation by scavenger compounds, the 5-hydroxy-4-keto site of naringin (i.e. held responsible for the enhancement of transition metal ion binding of flavonoids especially at neutral pH conditions [35]) may be more active than the 5-hydroxy-4-oxime site of naringin oxime which may exist in the form of syn- and anti- isomers, one of which being relatively less available for copper bonding. 3.8. Superoxide radical scavenging activity Superoxide anion (O 2 ) derived from dissolved oxygen by PMS– NADH coupling reaction reduces NBT in this system. In this meth2+ od, O 2 reduces the yellow dye (NBT ) to produce the blue formazan which is measured spectrophotometrically at 560 nm.
4. Conclusions In this study, naringin oxime was synthesized, characterized, and investigated for antioxidant and ROS scavenging activity. The oxime-compound of naringin was characterized on the basis of analytical and spectral data. The number and position of OH groups in the flavonoid structure have an important role on the antioxidant capacity of a flavonoid. Naringin oxime showed higher antioxidant capacity compared to pure naringin, as measured by the ETbased CUPRAC assay due to the preferential stabilization of 1e oxidized naringin oxime radical by intramolecular H-bonding. This suggests that the 4-oxime group (@NAOH) replacing 4-keto oxygen significantly changes the electron-transfer (ET) properties of naringin. On the other hand, the free radical (specifically hydroxyl and superoxide radicals) scavenging activities of naringin and naringin oxime were not so different possibly because the easy ET sites (5- and 40 -OH and 4-NOH) of naringin oxime are not necessarily the strongest free radical scavenger sites and because the mechanisms of ET and radical scavenging assays are different (i.e. radical quenching is less structure-specific than ET). As a result of this study, some new oxime-type antioxidants like naringin oxime can be derived by using different flavanones as starter materials, and these derivatives may potentially find use as a radioprotector and anticancer agents. Conflict of interest The authors declare no conflicts of interest. Acknowledgments Author Damla Akpınar would like to thank Istanbul University, Institute of Pure and Applied Sciences (I.U. Fen Bilimleri Enstitüsü), for the support given to her M.Sc. thesis entitled ‘‘Synthesis, Characterization and Investigation of Antioxidant Capacity of Some Flavanone-Oxime Compounds’’. The authors wish also to thank Assoc. Professor Eva Hellmén of the Department of Anatomy and Physiology, Uppsala University for kind donation of the CMT-U27 cell line and Dr. Fulya Üstün Alkan for anticancer activity research assistance. References
Fig. 7. Hydroxyl radical scavenging activity (inhibition vs concentration curve) of naringin oxime with respect to the modified CUPRAC method.
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Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
Novel oxime based flavanone, naringin-oxime: Synthesis, characterization and screening for antioxidant activity Mustafa Özyürek a,⇑, Damla Akpınar a, Mustafa Bener a, Baki Türkkan b, Kubilay Güçlü a, Resßat Apak a a b
Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar, Istanbul, Turkey Department of Chemistry, Faculty of Science and Letters, Harran University, Osmanbey, Sßanlıurfa, Turkey
a r t i c l e
i n f o
Article history: Received 31 July 2013 Received in revised form 3 December 2013 Accepted 29 January 2014 Available online 6 February 2014 Keywords: Naringin Naringin oxime Flavanone oximes Antioxidant activity Cupric reducing antioxidant capacity (CUPRAC) assay
a b s t r a c t Recent interest in polyphenolic antioxidants due to their involvement in health benefits has led to the investigation of new polyphenolic compounds with enhanced antioxidant activity. Naringin (40 ,5,7-trihydroxyflavanone-7-b-L-rhamnoglucoside-(1,2)-a-D-glucopyranoside) is one of the major flavanones in citrus and grapefruit. The present study aimed to synthesize naringin oxime from naringin and to evaluate its antioxidant and anticancer potential using in vitro assay system. The structure of the synthesized compound, naringin oxime, was elucidated by FT-IR, 1H NMR, elemental analysis and UV–vis spectroscopy. Antioxidant capacity of naringin oxime, as measured by the cupric reducing antioxidant capacity (CUPRAC) method, was found to be higher than that of the parent compound naringin. Other parameters of antioxidant activity (scavenging effects on OH, O 2 , and H2O2) of naringin and naringin oxime were also determined. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Flavonoids as an important group of natural substances have variable phenolic structures, and are found in abundance in fruits, vegetables, grains, flowers, wine, and tea [1]. They possess beneficial effects against serious diseases, such as cancer, cardiovascular disease, and neurodegenerative disorders [2]. In vitro experimental studies show that flavonoids act as antioxidants, antimicrobials, antivirals, and antiinflammatories [3]. Naringin (40 ,5,7-trihydroxyflavanone-7-b-L-rhamnoglucoside-(1,2)-a-D-glucopyranoside) is a member of the flavonoid family that shows various bioactivities on human health as antioxidant, reactive oxygen species (ROS) scavenger, antiinflammatory and antiapoptosis agent [4] having anti-carcinogenic [5] and neuro-protective effects [6], and especially a most investigated cancer preventive agent [7]. Naringin is found in abundance in citrus, grapefruit and juices [8]. When naringin is taken orally, it is metabolised by the enzymes a-rhamnosidase and b-glucosidase to its aglycone naringenine, which is in the more absorbable form [9,10], because naringin kinetically exhibits a delay in its intestinal absorption, resulting in decreased bioavailability. Naringin is a naturally available antioxidant that can be used for the synthesis of other novel antioxidants for ⇑ Corresponding author. Address: Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar, 34320 Istanbul, Turkey. Tel.: +90 212 473 7070x17627; fax: +90 212 473 7180. E-mail address: [email protected] (M. Özyürek). http://dx.doi.org/10.1016/j.cbi.2014.01.017 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
enhancing antioxidant activity. Pereira et al. synthesized a new naringin–metal complex, naringin–Cu(II), and this compound was found to possess higher antioxidant, antiinflammatory and tumor cell cytotoxicity activities than free naringin [11]. Oximes (Ox) (R1R2C@NOH) constitute an important class of hydroxylamines, where R1 is organic side chain and R2 is either hydrogen, forming an aldoxime, or another aromatic group, forming ketoxime [12]. Oximes and their derivatives are important intermediates in organic syntheses [13]. The oxime functional group can easily be bound to such important organic groups as carbonyl, amino, nitro and cyano functions and can also serve as a convenient protective group [14]. Oximes have been used for many important pharmaceutical and synthetic chemistry applications, and often act as chemical building blocks for the synthesis of agrochemicals and pharmaceuticals [12]. Oxime-type functional groups are included in many organic medicinal agents used in the treatment of organophosphate (OP) poisoning. Inhibition of acetylcholinesterase (AChE) results from acute OP toxicity, whereas oximes, by reactivating AChE, are considered to be rather effective against OP poisoning [15,16]. Therefore, synthesis and investigation of various oximes have an important role in medicinal research. Puntel et al. investigated the capacity of butane-2,3-dionethiosemicarbazone oxime to scavenge different forms of reactive species (RS) both in vivo and in vitro, and found significant hydrogen peroxide (H2O2), nitric oxide (NO) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity for the oxime [16]. The antiradical and
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antioxidant activities of four biologically active N,N-diethyloaminoethyl ethers of flavanone oximes were investigated by Metodiewa et al., and these compounds were shown to act as promising antioxidants and radioprotectors comparable to rutin activities under oxidative stress conditions [17]. Naringenin oxime was investigated for its antioxidant capacity by using the cupric reducing antioxidant capacity (CUPRAC) method, where the oxime functional group significantly enhanced the antioxidant capacity of pure naringenin [18]. Because of the important advantageous uses of oximes, especially of flavanone oximes, we synthesized naringin oxime (Fig. 1) and determined its antioxidant properties by using the CUPRAC method [19]. The chromogenic oxidizing reagent of the CUPRAC assay, bis(2,9-dimethyl-1,10-phenanthroline)copper(II) (abbreviated as Cu(II)-Nc), is simple, diversely applicable to both hydrophilic and lipophilic antioxidants, stable and easily available at low cost [19]. The CUPRAC method has been successfully applied to the determination of antioxidants in food plants (apricot, herbal teas, wild edible plants, herby cheese, etc.), natural dyes [20], and to human serum [21]. The main CUPRAC method was modified for measuring the hydroxyl radical scavenging activities of polyphenolics [22], xanthine oxidase (XO) inhibition activity [23], hydrogen peroxide scavenging activity of polyphenolics in the presence of Cu(II) catalyst [24], and development of a CUPRACbased antioxidant sensor on a Nafion membrane [25]. The synthesis reaction of naringin oxime compound is shown in Fig. 2. The antioxidant activities (scavenging effects on OH, O 2 , and H2O2) of naringin oxime have also been investigated. Scavenging activities against these reactive species were tested, because OH generated in the human body shows the strongest oxidative activity among ROS, H2O2 as a non-radical oxidizing species may be generated in tissues and diffuse across biological membranes, and O 2 has a great importance as a physiological signaling molecule while its scavengers can combat against many types of diseases [26,27]. The chelating ability toward transition metal ions is an important mechanism of secondary antioxidant action, because substitution of a C-4 (@O) group in the parent compound structure with a C-4 (@NAOH) group in the oxime derivative may enhance the antioxidant activity of the product through stronger binding of transition metal ions [18], thereby preventing Fenton-type reactions of reactive species generation. Further research is needed to show the possible pharmacological and biological activity of this newly synthesized naringin oxime compound, and this work is believed to potentiate the development of some new oxime-based antioxidants with enhanced antioxidant activity. 2. Materials and methods 2.1. Materials and apparatus All reagents and solvents were of analytical reagent grade. Naringin (40 ,5,7-trihydroxyflavanone-7-rhamnoglucoside) and
neocuproine (2,9-dimethyl-1,10-phenanthroline) were purchased from Sigma Chemical Co. (Steinheim, Germany). Copper(II) chloride dihydrate, ammonium acetate (NH4Ac), absolute ethanol (EtOH), methanol (MeOH), dimethyl sulfoxide (DMSO), hydroxylamine hydrochloride, sodium acetate trihydrate were purchased from Merck (Darmstadt, Germany). All chemicals used in reagent preparation were weighed with an accuracy of ±0.0001 g. The spectra and absorption measurements were recorded in matched Helma quartz cuvettes using a Varian CARY Bio 100 UV–vis spectrophotometer. The elemental analyses were performed using a CHNS instrument model Carlo-Erba 1106 elemental analyzer. 1H NMR spectra in DMSO were obtained on a Varian INOVA 500 MHz NMR spectrometer. The IR spectra were obtained by using KBr pellets in the spectral range 4000–400 cm1 on a Perkin Elmer RXI FT-IR spectrometer. Melting points were found with a Stuart Scientific SMP3 Melting Point Apparatus. 2.2. Preparation of solutions The standard solutions of naringin oxime and naringin were prepared in EtOH at a concentration of 1.0 mM. All standard solutions were stored at +4 °C prior to analysis. The CuCl2 solution (10.0 mM) and ammonium acetate buffer solution (1 M, pH 7.0) were prepared in distilled water and neocuproine solution (7.5 mM) in absolute ethanol. 2.3. Cell culture and treatment The canine mammary carcinoma cell line CMT-U27 (a generous gift from Assoc. Professor Eva Hellmén) was obtained from Uppsala University, Sweden. CMT-U27 cells were cultured in DMEM-F12 (Sigma Chemicals, St. Louis, USA), supplemented with 10% fetal bovine serum (Biological Industries, Israel), 100 IU mL1 penicillin G, 100 lg mL1 streptomycin, and 2.5 lg mL1 amphotericin B (Sigma, St. Louis, USA), at 37 °C in a humidified 5% CO2 atmosphere. Stock solution of naringin oxime was freshly prepared in DMSO and diluted with DMEM-F12 to a final concentration 60.1% of DMSO. Control groups received DMSO vehicle at a concentration equal to that in naringin oxime treated cells. 2.4. Synthesis of naringin oxime Naringin oxime was prepared by treating 0.01 mol (5.8 g) naringin with hydroxylamine hydrochloride (0.01 mol, 0.69 g) in 25 mL ethanol and sodium acetate trihydrate (0.01 mol, 1.36 g) in water. The solution was heated at reflux on a water bath for 4 h with constant stirring. On cooling to room temperature and adding 10 mL dichloromethane to solutions, a white solid precipitated out. It was filtered, washed with water, and dried over P2O5 under vacuum. The product yield was 60%. Melting point: 203 °C. Elemental OH
OH HO H3C
HO
OH O
O O 7
HO HO HO
O CH2
1
8
6
9 O 2 10
5 OH
Naringin
2'
O
4
3
1'
3'
'
6
4' 5'
OH
H3C
OH O
O O 7
HO O
HO HO
CH2
6
8
1 9 O 2
1'
10 4 3 5 OH N
Naringin oxime Fig. 1. The structures of naringin and naringin oxime.
2'
OH
3' 4' OH 6'
5'
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HO
HO
O
O
O
OH
NH 2OH. HCl /CH3COONa/MeOH
OH
4-5 h, rf
O
O
O HO
O
O
HO
OH HO
O HO HO
OH
N
OH
CH3
O OH
HO
O HO HO
OH
CH3
Fig. 2. Schematic representation of the synthesis of naringin oxime.
analysis results were; found (%): C 54.19; H 5.20; N 2.23; calculated for [C27H33NO14] [atomic mass 595.55]: C 54.45; H 5.59; N 2.35%. 2.5. Antioxidant capacity of naringin oxime measured by normal CUPRAC measurement The CUPRAC method is based on the reduction of light-blue colored cupric neocuproine complex (Cu(II)–Nc) by antioxidants to the yellow-orange colored cuprous chelate (Cu(I)–Nc) [19]. To a test tube were added 1.0 mL each of Cu(II), Nc, and NH4Ac buffer solutions. Antioxidant standard solutions (x) mL and H2O (1.1x) mL were added to the initial mixture so as to make the final volume (4.1 mL). The tubes were stoppered, and after 30 min standing at room temperature, the absorbance at 450 nm (A450) was recorded against a reagent blank. The standard calibration curves of each antioxidant compound was constructed in this manner as absorbance vs concentration, and the CUPRAC molar absorptivity of each antioxidant was found from the slope of the calibration line concerned. The scheme for normal measurement of antioxidants is summarized as: 1.0 mL Cu(II) + 1.0 mL Nc + 1.0 mL NH4Ac buffer + (x) mL antioxidant + (1.1x) mL H2O; total volume = 4.1 mL, measure A450 against a reagent blank after 30 min of reagent addition. 2.6. Incubated sample measurement by the CUPRAC method The mixture solutions containing sample and reagents were prepared as described in ‘normal measurement’; the tubes were stoppered and incubated for 20 min in a water bath at a temperature of 50 °C. The tubes were cooled to room temperature under running water, and their A450 values were measured [19]. 2.7. Hydroxyl radical scavenging (HRS) activity Hydroxyl radicals (OH) in aqueous media were generated through the Fenton system and spectrophotometrically determined via hydroxylation of a salicylate probe by the modified CUPRAC method [22]. To a test tube were added 1.5 mL of phosphate buffer (pH 7.0), 0.5 mL of 10 mM sodium salicylate, 0.25 mL of 20 mM Na2–EDTA, 0.25 mL of 20 mM FeCl2 solution, (2.0x) mL H2O, (x) mL naringin oxime solution (x varying between 0.1 and 0.5 mL) at a concentration of 2.0 105 M, and 0.5 mL of 10 mM H2O2 rapidly in this order. The mixture in a total volume of 5.0 mL was incubated for 10 min in a water bath kept at 37 °C. At the end of this period, the hydroxyl radical generation reaction was stopped with adding 0.5 mL of 268 U mL1 catalase solution,
and vortexed for 30 s. Final mixtures (0.5 mL of the incubation solution) were subjected to the HRS-CUPRAC method [22].The inhibition ratio of naringin oxime (%) was calculated using the equation:
Inhibition ratio ð%Þ ¼ 100
A0 A A0
ð2:1Þ
where A0 and A are the CUPRAC absorbances of the system in the absence and presence of scavenger, respectively. 2.8. Hydrogen peroxide scavenging (HPS) activity The ability of naringin oxime to scavenge hydrogen peroxide was determined according to the method of Özyürek et al. [24]. To a test tube were added 0.7 mL of phosphate buffer (pH 7.4), 0.4 mL of 1.0 mM H2O2, 0.4 mL of 0.1 mM CuCl22H2O in this order (H2O2 incubation solution, used as reference). To the other two test tubes were added 0.5 mL of phosphate buffer (pH 7.4), 0.4 mL of 1.0 mM H2O2, 0.2 mL naringin oxime solution, and 0.4 mL of 0.1 mM CuCl22H2O solution rapidly in this order (named as scavenger solutions-I and II; scavenger solutions-I and II, identical at this stage). The mixtures in a total volume of 1.5 mL were incubated for 30 min in a water bath kept at 37 °C. At the end of this period, to both reference and scavenger solution-I was added 0.4 mL H2O, and to scavenger solution-II was added 0.4 mL of 268 U mL1 catalase solution (the contents of scavenger solutionI and -II became different at this stage), and vortexed for 30 s. Final mixtures (1.0 mL of the incubation solution) were subjected to the HPS-CUPRAC method [24]. The HPS activity of naringin oxime (%) was calculated using the equation:
HPS ð%Þ ¼ 100
A0 ðA1 A2 Þ A0
ð2:2Þ
where A0 is the CUPRAC absorbance of reference H2O2 incubation solution, A1 and A2 are the CUPRAC absorbances of scavenger solutions-I and -II, respectively. 2.9. Superoxide anion radical scavenging activity The superoxide anion radicals (O 2 ) were generated in vitro in a non-enzymatic system (PMS-NADH) and determined spectrophotometrically by nitroblue tetrazolium (NBT) reduction method [28]. To a test tube were added (2.5x) mL DMSO, (x) mL (usually, x = 0.5) scavenger solution at a concentration of 1.0 103 M, 2.0 mL of 468 lM NADH, 1.0 mL of 300 lM NBT, in this order. The reaction was started by adding 1.0 mL of 60 lM PMS solution to the incubation mixture. The mixture in a total volume of 6.5 mL
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43
was incubated for 5 min in a water bath kept at 25 °C, and the absorbance was read at 560 nm against DMSO. Decreased absorbance of the incubation reaction mixture indicated increased superoxide anion radical scavenging activity. All experimental results were expressed as the mean ± standard deviation (S.D.) of triplicate determinations. The inhibition ratio of naringin oxime (%) was calculated using the equation:
Inhibition ratio ð%Þ ¼ 100
A0 A A0
ð2:3Þ
where A0 and A are the absorbances of the incubation reaction mixture in the absence and presence of scavenger, respectively. 2.10. Cell proliferation assay CMT-U27 cells were cultured in 96-well plates at a density of 1 104 cells/100 lL medium and allowed to attach for 24 h. Thereafter, medium was removed and replaced with 100 lL of medium containing various concentrations (0–1000 lM) of naringin oxime and incubated for 48 h. After incubation, cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) cell proliferation kit (Roche, Germany) [29]. Briefly, 10 lL of MTT solution 5 mg mL1 was added to each well and incubated for 4 h at 37 °C in CO2 incubator. The purple water insoluble formazan salt was then dissolved with 10% SDS in 0.01 M HCl and incubated overnight in a humidified 5% CO2 atmosphere. The optical densities (OD) of the wells were measured at 595 nm by microplate reader (Molecular Devices, USA). Cell proliferation inhibition rate (CPIR) was calculated using the following equation: CPIR, % = 100 [1 (average OD value of the treated wells)/(average OD value of the control wells)]. The effect of each compound on growth inhibition was assessed as percent cell viability where vehicle-treated cells were taken as 100% viable. 3. Results and discussion 3.1. Physical properties of naringin oxime The cream compound was stable at room temperature. It was soluble in MeOH, EtOH, and DMSO, but insoluble in water and DCM.
Fig. 3. UV–vis spectra of naringin and naringin oxime in ethanol.
3.3.1. Naringin 1 H-NMR (500 MHz, DMSO-d6): 12.32 (d, J = 3.25 Hz, 5-OH), 9.57 (s, 40 -OH), 7.31 (2H, m, H-20 and H-60 ), 6.79 (2H, dd, J = 1.49 and J = 1.39 Hz, H-30 and H-50 ), 6.10 (1H, brs, H-6), 5.49 (1H, m, H-2), 4.67 (1H, d, J = 4.64 Hz, H-3 ax), 4.60 (1H, d, J = 4.26 Hz, H-3 eq), 3.31 (1H, s, ACH2), 3.26 (1H, s, ACH2), 1.15 (3H, d, ACH3). 3.3.2. Naringin oxime 1 H-NMR (500 MHz, DMSO-d6): 12.03 (s, 5-OH), 11.34 (s, N-OH), 9.58 (s, 40 -OH), 7.32 (2H, m, H-20 and H-60 ), 6.79 (2H, d-d, J = 1.57 Hz, J = 1.34 Hz, H-30 and H-50 ), 6.10 (1H, t, H-6), 6.07 (1H, t, H-8), 5.49 (1H, m, H-2), 4.68 (1H, d, J = 4.57 Hz, H-3 ax), 4.61 (1H, d, J = 4.10 Hz, H-3 eq), 2.53 (1H, s, ACH2), 2.45 (1H, s, ACH2), 1.15 (3H, d, ACH3). The 1H NMR spectrum of naringin oxime exhibited very similar signals to that of naringin with very small chemical shift differences being in the interval 0.30–0.42 ppm for aromatic protons. There was an additional OH proton signal, observed at d 11.34, which was attributed to the oxime hydroxyl (C@NAOH). The presence of this hydroxyl was supported by the FT-IR spectrum (3395 cm1). 3.4. FT-IR study of naringin oxime
3.2. UV–vis spectroscopic study of naringin oxime The UV–vis spectra of naringin and naringin oxime in EtOH are shown in Fig 3. Naringin, like most flavones and flavonols, exhibits two major absorption bands in the UV region, at 284 nm (band I) representing B-ring absorption (cinnamoyl system), and at 229 nm (band II), considered to be associated with the absorption involving the A ring benzoyl system [30]. Compared to naringin absorption spectrum, band II of the naringin oxime compound is bathochromically shifted (i.e. to the longer wavelength region) as shown in Fig. 3, possibly due to added intra-molecular H-bonding alternatives between the C-4 (@NAOH) and C-5 (OH) substituents. Considering the greater hypsochromic shift of band I observed in the oxime derivative of naringenin relative to parent naringenin (i.e. from 294 nm of naringenin to 284 nm of naringenin oxime) [18], it can be deduced that the 7-b-L-rhamnoglucoside functionality of naringin reduced the spectral differences between naringin and naringin oxime.
The FT-IR spectra of naringin and naringin oxime are shown in Fig. 4. All the spectra are characterized by vibrational bands mainly due to the OH, CAH, C@O, C@N, and C@C functional groups. The t (OH), t (CAH), t (C@O), t (C@C) for naringin appear at 3425 cm1, 2957–2859 cm1, 1645 cm1, and 1597–1502 cm1, respectively. The t (OH), t (CAH), t (C@N), t (C@C) for naringin oxime appear at 3395 cm1, 2970–2875 cm1, 1620 cm1, and 1582–1520 cm1, respectively. The naringin band at 1645 cm1 may be assigned to t (C@O) stretching, whereas in naringin oxime, the band at 1620 cm1 assignable to t (C@N) confirms the presence of NAOH group bound to naringin. Türkkan et al., in reporting the synthesis and characterization of naringenin oxime, observed that the naringenin band at 1640 cm1 assigned to t (C@O) was shifted to 1628 cm1 assignable to t (C@N) when the oxime group was inserted to the main structure [18]. 3.5. Antioxidant capacity of naringin and naringin oxime measured by the CUPRAC method
3.3. 1H NMR study of naringin oxime The 1H NMR (DMSO) data of naringin and naringin oxime are separately given below:
The reaction equation for the CUPRAC method: Cu(II)–Nc with n-electron reductant phenolic antioxidants [19] can be formulated by:
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Transmittance (%)
(a)
4000
(b)
3500
3000
2500
2000
1500
1000
500
Wave Number (cm ) -1
Fig. 4. The FT-IR spectra of solid compounds: (a) naringin oxime; (b) naringin.
þ þ nCuðNcÞ2þ 2 þ ArðOHÞn $ nCuðNcÞ2 þ Arð@OÞn þ nH
ð1Þ
The CUPRAC method [19] was applied to naringin and naringin oxime as two interrelated procedures, i.e., normal (at room temperature) and incubated (at 50 °C) versions of the assay, in comparison with the trolox standard reference compound. The linear calibration equations of naringin oxime and naringin (as absorbance in a 1-cm cell vs molar concentration) gave the molar absorption coefficient (e) as the slope (Table 1); the linear working ranges over which Beer’s law was valid are given in the same table. The CUPRAC molar absorption coefficient of the tested antioxidant divided by that of trolox under the same conditions gave the (unitless) trolox equivalent antioxidant capacity (TEAC), or TEAC coefficient of that antioxidant (Table 1). Fig. 5 shows the CUPRAC reaction kinetics with individual antioxidants measured at room temperature. It is apparent from Fig. 5 that naringin and naringin oxime showed a gradual absorbance increase with time, which determined the time period of measurement (i.e., 30 min after the mixing of reagent with the analytes). As the reduction potential of the antioxidant approaches that of the reagent, the thermodynamic efficiency and possibly the rate of the oxidation reaction decreases, which is the case for naringin (Ered o = 0.6 V). The anodic peak potential of the first oxidation wave of naringenin (i.e. the aglycon of naringin) was reported as 0.688 V [31]. In the CUPRAC method, the limit of quantification (LOQ) for naringin and naringin oxime were determined to be 22.7 lM and 0.75 lM, respectively, enabling a much higher sensitivity for the latter. The calibration curves of naringin and naringin oxime (CUPRAC absorbances vs antioxidant concentrations) are shown in Fig. 6. All of the easily oxidized flavonoids exhibited standard reduction potentials of 60.2 V, whereas naringin, having a potential þ close to that of the CuðNcÞ2þ 2 CuðNcÞ2 couple [19], underwent a slow reaction with the reagent. Naringin oxidation could only be enhanced after 50 °C incubation in the CUPRAC method, but its TEAC coefficient still remained low (i.e. increased from 0.02 to
Fig. 5. CUPRAC reaction kinetics with individual antioxidants; rate of increase in absorbance at 450 nm for 1 mM solutions of naringin and naringin oxime (at room temperature).
0.13 with incubation, Table 1). Regarding the antioxidant efficiency of flavanones, Yuting et al. tested several flavonoids in the inhibition of lipid peroxidation in mouse liver homogenates, and found several micromolar IC50 values for rutin, morin, and quercetin, but no inhibition for the flavanones including naringin, confirming that the antioxidant activity of naringin is not of the same magnitude as of common flavonoids [32]. On the other hand, the TEAC coefficients of naringin oxime measured with the normal and incubated CUPRAC methods (TEACN = 0.52 and TEACInc = 4.70, Table 1) were found to be much higher than the corresponding values of naringin, probably as a result of increased stabilization of the 1-e oxidized product (i.e., nitroxyl-aryloxyl radical) of naringin oxime as compared to that of naringin by intra-molecular H-bonding [33]. As the reduction potential of the redox pair: {1e oxidized product/parent compound} is lowered in flavonoids due to stabilization of the aryloxyl radical, the antioxidant power of the concerned compound (in this case, naringin oxime) is enhanced [34]. 3.6. Hydroxyl radical scavenging (HRS) activity Hydroxyl radical (OH), which can easily react with amino acids, DNA and membrane components, shows the strongest chemical reactivity among ROS. HRS of naringin oxime as a function of lM concentration with respect to Eq. (2.1) using the modified CUPRAC method (HRS-CUPRAC) [22] is given in Fig. 7. Naringin oxime showed statistically (at 95% confidence level) similar HRS, the effect being (79.9 ± 1.6)% at the initial concentration of 20 lM, compared to that of naringin (81.5 ± 0.7)% at the same concentration (N = 3), and HRS activities were dose-dependent. Seemingly, the replacement of 4-keto functionality in naringin with 4-NOH in naringin oxime does not make a difference in the HRS activities of the two compounds, as HRS action is inherently less structurespecific than other similar assays of ROS quenching due to the very high rates of hydroxyl radical reactions with biological substrates at the second-order rate constants of 108109 M1 s1.
Table 1 The molar absorption coefficients, linear ranges and TEAC coefficients of naringin and naringin oxime with respect to the CUPRAC method (normal (N) and incubated (Inc.) CUPRAC methods). Antioxidant
Naringin Naringin oxime
Molar absorption coefficient e (L mol1 cm1) CUPRACN
CUPRACInc.
3.3 102 8.6 103
2.2 103 7.8 104
Linear range (mol L1)
1.5 104 – 3.7 103 6.3 106 – 1.5 104
TEACCUPRAC TEACN
TEACInc
0.02 0.52
0.13 4.70
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Antioxidants are able to inhibit formazan formation [28]. The decrease of absorbance at 560 nm with antioxidants indicates the consumption of O 2 in the reaction mixture. The percentage inhibition of O 2 by 1 mM concentrations of naringin oxime and naringin oxime were found as (6.8 ± 0.6)% and (4.8 ± 0.5)%, respectively. 3.9. The effects of naringin oxime on cell proliferation Cell viability was assessed by MTT assay based on the conversion of the water soluble MTT to an insoluble colored formazan product by the active mitochondria of living cells [29]. After 48 h incubation, naringin oxime inhibited cell proliferation at 100, 200, 400 and 1000 lM by 10.39%, 11.79%, 12.7% and 17.85% of the control level, respectively. However, IC50 value for naringin oxime was not observed in the concentration range studied, and the corresponding IC50 value could not be calculated.
Fig. 6. The calibration curves as absorbance vs concentration of naringin oxime and naringin (inset) with respect to the normal CUPRAC method.
3.7. Hydrogen peroxide scavenging (HPS) activity The ability of naringin oxime to scavenge hydrogen peroxide was measured by the HPS-CUPRAC method [24] and compared to that of naringin. Hydrogen peroxide scavenging (HPS) activity of naringin oxime at 1 mM (initial conc.) was found to be (3.3 ± 0.1)%. On the other hand, naringin exhibited (26.0 ± 1.2)% HPS activity at the same concentration (N = 3). Since HPS-CUPRAC is a transition metal ion- (specifically Cu(II)-) catalyzed assay involving H2O2 degradation by scavenger compounds, the 5-hydroxy-4-keto site of naringin (i.e. held responsible for the enhancement of transition metal ion binding of flavonoids especially at neutral pH conditions [35]) may be more active than the 5-hydroxy-4-oxime site of naringin oxime which may exist in the form of syn- and anti- isomers, one of which being relatively less available for copper bonding. 3.8. Superoxide radical scavenging activity Superoxide anion (O 2 ) derived from dissolved oxygen by PMS– NADH coupling reaction reduces NBT in this system. In this meth2+ od, O 2 reduces the yellow dye (NBT ) to produce the blue formazan which is measured spectrophotometrically at 560 nm.
4. Conclusions In this study, naringin oxime was synthesized, characterized, and investigated for antioxidant and ROS scavenging activity. The oxime-compound of naringin was characterized on the basis of analytical and spectral data. The number and position of OH groups in the flavonoid structure have an important role on the antioxidant capacity of a flavonoid. Naringin oxime showed higher antioxidant capacity compared to pure naringin, as measured by the ETbased CUPRAC assay due to the preferential stabilization of 1e oxidized naringin oxime radical by intramolecular H-bonding. This suggests that the 4-oxime group (@NAOH) replacing 4-keto oxygen significantly changes the electron-transfer (ET) properties of naringin. On the other hand, the free radical (specifically hydroxyl and superoxide radicals) scavenging activities of naringin and naringin oxime were not so different possibly because the easy ET sites (5- and 40 -OH and 4-NOH) of naringin oxime are not necessarily the strongest free radical scavenger sites and because the mechanisms of ET and radical scavenging assays are different (i.e. radical quenching is less structure-specific than ET). As a result of this study, some new oxime-type antioxidants like naringin oxime can be derived by using different flavanones as starter materials, and these derivatives may potentially find use as a radioprotector and anticancer agents. Conflict of interest The authors declare no conflicts of interest. Acknowledgments Author Damla Akpınar would like to thank Istanbul University, Institute of Pure and Applied Sciences (I.U. Fen Bilimleri Enstitüsü), for the support given to her M.Sc. thesis entitled ‘‘Synthesis, Characterization and Investigation of Antioxidant Capacity of Some Flavanone-Oxime Compounds’’. The authors wish also to thank Assoc. Professor Eva Hellmén of the Department of Anatomy and Physiology, Uppsala University for kind donation of the CMT-U27 cell line and Dr. Fulya Üstün Alkan for anticancer activity research assistance. References
Fig. 7. Hydroxyl radical scavenging activity (inhibition vs concentration curve) of naringin oxime with respect to the modified CUPRAC method.
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