Radical scavenging propensity of Cu2 +, Fe3 + complexes of flavonoids and in-vivo radical scavenging by Fe3 +-primuletin

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 171 (2017) 432–438

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Radical scavenging propensity of Cu2 +, Fe3 + complexes of flavonoids and in-vivo radical scavenging by Fe3 +-primuletin Erum Jabeen a, Naveed Kausar Janjua a,⁎, Safeer Ahmed a, Iram Murtaza b, Tahir Ali b, Shahid Hameed a a b

Department of Chemistry, Quaid-i-Azam University, Islamabad -45320, Pakistan Department of Biochemistry, Quaid-i-Azam University, Islamabad -45320, Pakistan

a r t i c l e

i n f o

Article history: Received 3 May 2016 Received in revised form 17 August 2016 Accepted 17 August 2016 Available online 22 August 2016 Keywords: Metal-flavonoid complexes Radical scavenging DPPH• TEMPO• Superoxide anion radical Hydroxyl free radical

a b s t r a c t Cu2+ and Fe3+ complexes of three flavonoids (morin or mo, quercetin or quer and primuletin or prim) were synthesized with the objective of improving antioxidant capacities of flavonoids. The radical scavenging activities of pure flavonoids and their metal complexes were assayed to monitor their tendencies towards sequestering of radicals at physiological conditions. The scavenger potencies of metal-flavonoid complexes were significantly higher than those of the parent flavonoids. Further, influence of the solvent polarity on the radical capturing by flavonoids and their metal complexes was in favor for the polar solvent. Fe3+-prim displayed its radical scavenging ability via up gradation of CAT and SOD activities in in-vivo antioxidant assays. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Radical scavenging activity possessed by flavonoids make them highly debated class of compounds. Resonance stabilized structure of flavonoids can quench highly reactive free radicals converting them to less reactive aroxyl radicals, thus lending flavonoids a rank of antioxidant compounds. Flavonoids are antioxidants not only because of their radical scavenging ability but also due to their power to chelate metal ions [1–3]. They act as strong chelator towards metal ions and can form different complexes [4]. It is well reported in literature that metal chelated flavonoids exhibits all the properties of free flavonoids but their pharmacokinetic features are enhanced upon complexation by the metal ion [5]. Some chelated flavonoids are reported to be much more effective scavenger of free radicals than the free flavonoids. For example, De Suza et. al. have reported the complexes of rutin, quercetin and galanin with aluminum(III) and zinc(II) to have greater free radicals scavenging capability than free flavonoid [6]. Pt(II)- and Pd(II) chelation of morin enhanced radical scavenging effect [7]. Maleˇsev and Kunti [8] showed that UO2–rutin, complexes have advanced properties than their free flavonoid counterparts. Therefore, metal chelated

Abbreviations: mo, morin; quer, quercetin; prim, primuletin; DPPH•, 1,1-dipheny-l-2picryhydrazyl; Tempo•, 2,2,6,6-tetramethylpipyridin-1-yloxy radical; Fls, flavonoids; mFls, metal-flavonoids; CV, cyclic voltammetry. ⁎ Corresponding author. E-mail address: [email protected] (N.K. Janjua).

http://dx.doi.org/10.1016/j.saa.2016.08.035 1386-1425/© 2016 Elsevier B.V. All rights reserved.

flavonoids constitute the current research topic towards drug development [9]. The pharmacological benefits arise due to the antioxidant activity of flavonoids [10]. Being permanent part of our food, they suppress the oxidants present/formed in the body by virtue of their structural properties. This suppression of oxidants occurs via electron donation from free hydroxyl radicals on the flavonoid converting them to less reactive radicals, thus playing only a moderate role in propagation of radical induced damage in biological systems [11]. The enhancement of pharmacokinetic abilities upon chelation can be attributed to acquisition of extra dismutating center in complexed metal flavonoids providing them with better radical scavenging ability [12]. So it can be expected that greater number of flavonoids attached to metal atoms might contribute to better radical scavenging potencies. Therefore in the present work, Cu and Fe complexes of flavonoids (m-Fls) are prepared in 1:2 and 1:3 m:Fl and their antioxidant activity is studied in comparison with those of free flavonoids against 1,1-dipheny-l-2-picryhydrazyl (DPPH•), 2,2,6,6-tetramethylpipyridin1-yloxy radical (Tempo•), hydroxyl free radical (OH•) and superoxide radical (O•− 2 ) using various techniques. Further, flavonoids are practically soluble in organic solvents only and insoluble in water. In one of our previous work, efforts were made to solubilize metal-flavonoid complexes in water through inclusion complexation [13] but that pathway is expected to protect flavonoids against rapid oxidation by free radicals thus, misleading the quenching ability of flavonoids [14]. In the present work, three flavonoids are solubilized in different ratios of aqueous/organic (water/methanol) solvents

E. Jabeen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 171 (2017) 432–438

433

and modulating effect of solvent on antioxidant activity of metalflavonoid complexes was also detailed.

In superoxide assay, performed by the method of A.V. Kostyuk et al., absorbance of sample and blanks were used to calculate RSA [16]:

2. Experimental

O2•− scavenging activity ð%Þ ¼

ΔA b −ΔAs  100 ΔA b

ð4Þ

2.1. Apparatus Absorption spectra were recorded on Shimadzu 1601 spectrophotometer equipped with a Julabo F-34 thermostat (±0.1⁰C) using a pair of 1 cm path length quartz cuvette. Cyclic voltammetry (CV) was performed on Gamry electrochemical system 370 using three electrode cell assembly consisting of SCE reference electrode (sentek/ UK Company Cat # 924,005), Pt counter electrode and glassy carbon working electrode of 2.0 mm diameter. DFT calculations were run using ADF software package 2010.

A0 εG εG 1 ¼ þ : A−A0 εH−G −ε G εH−G −ε G K scav ½Fl or m−Fl

2.2. Stoichiometry and preparation of metal flavonoid complexes Cu2 + and Fe3 + complexes of three flavonoids quercetin (quer), morin (mo) and primuletin (5-hydroxyflavone or prim) were synthesized by the reported method [15]. Job's method was employed for the determination of molar ratio of metal and flavonoid in the synthesized complexes spectrophotometrically.

The radical scavenging activity assay was carried out as described by Jabbri et al. [11] and the RSA values were evaluated using the following equation for metal-flavonoid complex (m-Fl):    A −AS  100 RSA ð% Þ ¼ 1− mix A0

ð1Þ

RSA value of pure flavonoid (Fl) was calculated by the equation: A0 −Amix A0

  100

ð2Þ

In cyclic voltammetry, the variation in peak currents of Fls/m-Fls with added radical was used to evaluate the RSA value relative to the peak current of the metal flavonoid complexes as blank using the following equation; i0 −imix i0

  100

ð3Þ

0.30

log

1 Ip þ logK scav ¼ log Ip0 −Ip ½Fl or m−Fl

ð6Þ

ΔG ¼ −RTln K scav

logðRSAÞ þ log½H2 O ¼ a:1=εñb

Cu-prim

0.10

ð8Þ

All the experiments were performed in triplicate and the data was averaged. 2.4. In-vivo antioxidant studies of Fe3+-prim Sprague-Dawley rats were divided into four groups (n = 3 in each group, each rat weighing between 160 and 200 g). Group I (alloxan + Fe3 +-prim) was administrated with a single injection of alloxan; after induction of diabetes, the rats were given daily

0.5

Cu-mo

0.20

ð7Þ

All the experiments were done under physiological conditions of temperature and pH (310 K and pH -7.4 in PBS). DFT calculation were run on ADF 2014/08 software package using ATZ2P basis set at hybrid B3LYP SCF potential for Fl/m-Fls and their radicals generated as a result of their antioxidant activities. For solvent effect assay, the antioxidant activity was calculated in different ratios of water-methanol mixtures according to Yasuda– Shedlovsky approach [19,20], from the plot of log (RSA) + log [H2O] versus 1/ε:

Fe-quer

Cu-quer

Absorbance

 RSAð% Þ ¼

And from cyclic voltammetry, using following equation;

Absorbance



Fe-prim

0.4

Fe-mo 0.3 0.2 0.1

0.00

(a)

ð5Þ

ΔG was calculated form Kscav using [19];

2.3. Radical scavenging activity

RSAð% Þ ¼

Superoxide anion radical was generated electrochemically in DMSO containing 0.1 M TBAP at a scan rate 20 mV/s in potential window of − 1.0 V – 0.0 V and volumetric titrations of generated superoxide were done with antioxidants to evaluate RSA [17]. To avoid metal chelation by the free flavonoids, metal-free Fenton system described by Nappi was employed and the absorbance variation at 534 nm was recorded as a function of RSA of OH• [18]. The scavenging constants were calculated from UV–Vis spectroscopic data using;

0.0 0.0

0.3 XCu 2+

0.6

0.9

(b)

0.0

0.3

XFe 3+

Fig. 1. Jobs plots revealing maxima at (a) XCu2+ = 0.3 and (b) XFe3+ = 0.25.

0.6

0.9

434

E. Jabeen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 171 (2017) 432–438

prim

1.8

Fe-prim

1.8

0 uM

0uM

40uM

36uM

1.2

71uM 73uM 76uM 98uM

0.6

63uM

Absorbance

Absorbance

65uM

112uM

1.2

58uM 65uM 89uM 99uM

0.6

107uM

121uM

121uM

0.0

(a)

0.0 250

450

650

850

wavelength (nm)

prim

i (µA)

650

850

Fe-prim

0uM

0uM

36uM

40uM

53uM 65uM 85uM

0.3

450

wavelength (nm)

1.5

i (µA)

0.9

250

(b)

60uM

0.9

65uM 70uM

97uM 101uM

75uM 0.3

80uM

114uM -0.3

-0.3 0.2

(c)

90uM

0.4

0.6

0.8

E (V) vs.SCE (3 M)

0.2

0.4

(d)

0.6

0.8

E (V) vs.SCE (3 M)

Fig. 2. Effect of incremental addition of (a) prim (b) Fe-prim on UV–Vis spectrum of DPPH• at pH -7.4 (PBS) and 310 K and of (c) prim (d) Fe-prim on the cyclic voltammograms of DPPH• at pH -7.4 (PBS) at 0.1 V/s and 310 K.

intraperitoneal (i.p) injections of Fe3+-prim (140 mg/kg) for 15 days. Group II (Fe2+-prim) was administrated with daily injections of Fe3+prim for 15 days. Group III (alloxan) received a single dose of alloxan (140 mg/kg). Group IV (control) received daily injections of normal saline (1.2 ml) for due days. At the end of 15 days, all the experimental animals were sacrificed and serum was assayed for total ROS unit, CAT activities and SOD activities by reported methods using UV–Vis spectroscopy [21–23]. 3. Results and discussion 3.1. Stoichiometry of M-Fls Jobs method of continuous variation revealed that maxima was obtained at XCu2 + = 0.3 (Fig. 1(a)) and at XFe3 + = 0.25 (Fig. 1 (b)). Thus in Cu complexes of quer, mo and prim the metal to ligand ratio is 1:2. This means that two ligand molecules are attached with each Cu2+ ion to form a complex while Fe3+ complexes have metal to ligand ratio of 1:3. Thus in Fe-prim, Fe-mo and Fe-quer, three flavonoid molecules are attached per Fe3+ ion. 3.2. Antioxidant activity

(m-Fls) upon incremental addition at 0.1 V/s and 310 K, as shown in Fig. 2 (c and d) for prim and Fe3+-prim, provides a clear evidence of interaction between complex and the radical. The radical scavenging activity of 1.25 × 10−5 mol/dm3of flavonoids and their Cu2+ and Fe3+ complexes are reported in Table 1. The results obtained through electrochemical analysis complimented results of photometric analysis. It is clear from Table 1 that radical scavenging ability of quer is greater than that of mo which in turn, has higher antioxidant power than prim. RSA of metal complexes of flavonoids is greater than that of respective free flavonoid no matter which metal ion is being chelated by flavonoid. Based on these results, it can be inferred that complexation has raised the radical scavenging capacity of flavonoids. This trend can be attributed to the acquisition of extra dismutating center in complexed metal flavonoids providing them with better radical scavenging ability. Table 1 Scavenging activity data of flavonoids and their metal complexes obtained through UV– Vis spectroscopy and cyclic voltammetry. RSA (%) from Uv using Complex 2+

Stable free radicals DPPH• and Tempo• and highly reactive free radi• cals O•− 2 and OH were used to evaluate radical scavenging ability of flavonoids. The absorbance of both DPPH• decreased on incremental addition of any one of flavonoids or their complex as shown in Fig. 2 (a and b). The shift in peak potentials and/or current decay in cyclic voltammograms of DPPH• caused by either flavonoids (Fls) or their metal complexes

Cu -prim Fe3+-prim Cu2+-mo Fe3+-mo Cu2+-quer Fe3+-quer prim mo quer

RSA (%) from CV using

DPPH• Tempo• O-• 2

OH•

DPPH• Tempo• O-• 2

OH•

12.10 7.91 24.95 20.95 33.55 36.81 5.60 11.65 21.55

50.40 31.57 79.11 85.62 94.69 100.90 26.37 36.34 61.88

9.99 7.10 24.95 20.30 33.45 35.90 5.81 12.01 21.87

48.89 31.21 76.22 82.89 93.28 102.10 26.26 34.75 61.27

9.15 4.60 26.75 24.65 31.35 32.30 4.31 10.60 19.20

39.06 26.37 61.88 65.79 72.67 100.00 22.32 29.90 50.01

8.55 4.45 26.05 24.65 32.15 33.35 4.36 10.82 19.28

38.58 26.82 61.27 62.50 73.53 100.80 22.24 29.21 49.60

E. Jabeen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 171 (2017) 432–438

80

with DPPH˙

70

70

60

60

RSA (%)

RSA (%)

80

50 40

with Tempo˙

50 40

Fe-prim Fe-mo Fe-quer prim quer

30 20

Cu-prim Cu-mo Cu-quer mo

Fe-prim Fe-mo Fe-quer prim quer

30 20

10

(a)

435

Cu-prim Cu-mo Cu-quer mo

10 5

25

45

65

85

105

125

(b)

[Fl/m-Fl] (uM)

5

25

45

65

85

105

125

[Fl/m-Fl] (uM)

100

with O2- •

with OH˙

90

RSA (%)

RSA (%)

80

60

40

50

30

20 Fe-prim Cu-mo prim

0

(c)

70

0.5

1.0

Cu-prim Fe-quer mo

Fe-mo Cu-quer quer

1.5

Fe-prim Cu-mo prim

10

2.0

(d)

[Fl/m-Fl] (uM)

0.5

1.0

Cu-prim Fe-quer mo

Fe-mo Cu-quer quer

1.5

2.0

[Fl/m-Fl] (uM)

• Fig. 3. Variation of RSA with concentration of flavonoid-metal complexes against (a) DPPH•, (b) Tempo• (c) O•− 2 and (d) OH .

Further evaluation of data in Table 1 revealed that Cu2+ complexes of mo and prim exhibited better antioxidant power against DPPH• and Tempo• than their Fe3+ complexes while in case of quer, the sequence reverts. Fe3+ complex of quer scavenges both radicals more readily than Cu2+-quer complex. Fe3+ complexes of mo and quer scavenges compar2+ comatively more reactive OH• and O•− 2 radicals better than their Cu plexes while Cu2+ complex of prim exhibit higher RSA than Fe3+-prim. UV–Vis data of RSA (%) plotted as a function of concentration of flavonoids and their Cu2+ and Fe3+ complexes, was used to calculate IC50 corresponding to 50% RSA (Fig. 3). IC50 is the amount of scavenger required to consume 50% of free radical. It is inversely related to antioxidant activity of scavenger. The IC50 of flavonoids were found to in order of quer b mo b prim with DPPH• used, Table 2. Thus, the antioxidant activity of these flavonoids increased in order quer N mo N prim. and OH• were The same trend was obtained when Tempo•, O•− 2 employed as radical systems. In case of metal-flavonoid complexes, the IC50 values of both Cu2+ and Fe3+ complexes of quer, mo and prim were less than those of respective flavonoids, no matter which radical was used for analysis. Considering relative activity of Cu2+ and Fe3+ complexes of flavonoids, the

IC50 trend is same as that of RSA (%) reported in Table 1. The overall order of IC50 against Tempo• and DPPH• was: Fe3+-prim N Cu2 +-prim N Fe3+-mo N Cu2+-mo N Cu2+-quer N Fe3 +quer While versus OH• and O•− 2 , it was: Fe3+-prim N Cu2 +-prim N Cu2 +-mo N Fe3+-mo N Cu2+-quer N Fe3 +quer The trend of antioxidant activity would be opposite to that of IC50. The slight difference in these trends among the radicals can be attributed to small size and high reactivity of small free radicals OH• and O•− 2 as compared to DPPH• and Tempo•. 3.3. DFT calculations for radical scavenging activity The expected radical scavenging activity of the compounds was predicted based on the relative ΔEHomo (difference between the EHomo of

Table 2 IC50 values of flavonoids and their metal complexes. IC50 values (μM) DPPH•

Tempo•

OH•

O•− 2

Flavonoids

Pure

Cu(II)

Fe(III)

Pure

Cu(II)

Fe(III)

Pure

Cu(II)

Fe(III)

Pure

Cu(II)

Fe(III)

quer mo prim

21 34 81

17 25 50

15 29 78

20 31 84

18 22 52

13 26 77

1.25 2.09 2.80

0.86 1.01 1.60

0.64 0.95 2.37

1.01 1.72 2.37

0.66 0.79 1.24

0.62 0.73 1.98

436

E. Jabeen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 171 (2017) 432–438

Table 3 ΔEHomo and I.P. of the flavonoids and their metal-complexes calculated for most stable structures. ΔEHOMO (Hartree)

I.P. (kcal/mol)

Ligand

Free ligand

Cu2+-ligand 1:2

Fe3+-ligand 1:3

Free ligand

Cu2+-ligand 1:2

Fe3+-ligand 1:3

Prim mo quer

-0.015 -0.027 -0.035

-0.084 -0.092 -0.127

-0.103 -0.117 -0.152

172.34 168.54 166.95

157.24 151.23 148.79

153.64 147.65 141.23

radical generated as result of radical scavenging and EHomo of respective compound) and IP for the Fls and m-Fls (using Hartree approximation). The negative value of ΔEHomo shows that the HOMO of radical is comparatively more stable than the HOMO orbital of respective compound, Table 3. The more negative is ΔEHomo, more stability is achieved upon radical formation from the compound, and thus greater will be ability to scavenge the free radicals. Table 3 reveals that the ionization potentials of flavonoids (ligand) were more than those of their metal complexes which points to the fact that it is easy for the complex to donate the electrons as compared to the free ligands. This means that the complex is predicted to be better mediator for the electron transfer. The trend of ΔEHomo also discloses that HOMO orbital gains more stability upon radical formation in case of Fe3+ − flavonoid complex than either Cu2+-flavonoid complex or free flavonoid. Thus, the DFT calculations predicted that among flavonoids and their metal complexes, Fe3+ complexes are comparatively better radical scavenger than Cu2+-complexes which in turn are better than free ligands. Therefore, overall relative predicted trend of radical scavenging by the flavonoids and their complexes may be of the order: Fe3 +-quer N Cu2 +-quer N Fe3 +-mo N Fe3+-Prim N Cu2+-mo N Cu2+Prim N quer N mo N prim Fig. 4 depicts different possible sites for H atom transfer during radical scavenging. Table 4 reveals data corresponding to predicted ΔG (calculated for HAT mechanism) at different sites in free flavonoids and their metal complexes for OH• and O•− 2 scavenging. Site R5 corresponding to 4′-OH in structure of mo and quer can scavenge both OH•

and O•− 2 more spontaneously than any other site in these flavonoids. For Cu2+-prim and Fe3+-prim, the predicted mechanism will be electron transfer as there is no available OH moiety. It is also revealed in Table 4 that R1 which is not available in metal complexes of mo and quer is not susceptible to scavenge radical in free flavonoid as DFT predicted radical attack at R1 to be non-spontaneous in terms of positive ΔG. The predicted trend of flavonoids and their metal complexes towards radical scavenging of all four radical predicted by DFT calculation of free energy is as under; Fe3+-quer N Cu2+-quer N Fe3+-mo N Cu2+-mo N quer N Cu2+-prim N Fe3+-prim N mo N prim DFT calculations predicted that all the four radicals would be scavenged in the same order by the flavonoids. Table 5 reveals the comparison of computational and experimental data in terms of ΔG values. DFT calculations and results from U.V.-Vis and C.V. are in good agreement with each other revealing that DFT predictions of most plausible cite worked well. The ΔG values calculated through DFT calculation compliment the experimental value with slight difference. Further, the metal-flavonoids complexes were predicted to scavenge the free radicals more readily through DFT calculations. The DFT predictions were verified experimentally. This is in agreement with some literature where 1:1 Cu-morin has better radical scavenging than morin [15] and 2:1 Cu-quer has better scavenging properties than quer [24] against DPPH•. Cu-quer and Fe-quer with superoxide scavengers (%) in xanthine/ hypoxanthine [5] system is reported to have more antioxidant potential as depicted under physiological conditions in saline buffer environment in the present work.

3.4. Bond dissociation enthalpies for Fls and m-Fls Bond dissociation enthalpies are reported in Table 6. The computed bond dissociation enthalpies are b 85 kcal/mol for most reactive sites in Fls and m-Fls which is in agreement with literature reported for dihydroxy flavones [25,26]. Further Bond dissociation enthalpies are less for metal-flavonoids as compared to flavonoids which make metalflavonoids more active radical scavenger than flavonoids [27].

Fig. 4. Possible sites of attack by radicals in mo, quer and prim.

E. Jabeen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 171 (2017) 432–438

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Table 4 ΔG for interaction of OH• and O•− 2 at different sites on flavonoids and their metal complexes. ΔG (kJ/mol) for interaction of radical with flavonoid at R site OH•

O•− 2

Ligand

Site in ligand

Free Flavonoid

Cu2+ complexes

Fe3+ complexes

free Flavonoid

Cu2+ complexes

Fe3+ complexes

prim

Ro Ret R1 R2 R3 R4 R5 R1 R2 R3 R4 R5

20.8 – -3.1 -1.2 4.3 4.2 22.5 -4.7 -2.2 4.1 3.7 23.9

– 23.1 – 3.3 6.3 6.3 24.3 – -4.6 5.1 5.3 25.4

– 22.5 – -4.5 4.1 4.0 25.1 – -5.1 1.3 1.3 26.7

20.4 – -3.0 -1.5 3.1 1.2 22.0 -3.5 -5.8 2.3 1.5 23.1

– 22.9 – -5.7 4.1 4.2 23.5 – -4.3 4.0 4.1 24.8

– 22.4 – -6.7 2.3 3.3 24.5 – -4.4 4.0 2.4 25.1

mo

quer

Table 5 Comparison of -ΔG (kJ/mol) for all systems. -ΔG (kJ/mol) for scavenging of radicals by flavonoid calculated from different techniques DPPH•

Tempo•

OH•

O•− 2

Flavonoid

DFT

UV

CV

DFT

UV

CV

DFT

UV

CV

DFT

UV

CV

Prim Cu2+-prim Fe3+-prim mo Cu2+-mo Fe3+-mo quer Cu2+-quer Fe3+-quer

19.3 21.8 21.3 20.9 22.4 23.4 22.1 23.7 24.0

24.3 23.2 19.3 23.9 19.0 19.4 23.6 22.4 20.3

25.5 21.7 22.1 23.4 23.0 19.9 22.0 23.7 22.9

19.7 22.0 21.4 21.4 23.2 24.0 22.8 24.3 25.6

23.8 24.2 19.7 23.8 22.6 21.7 25.4 23.3 24.5

23.6 23.2 20.6 23.5 18.1 20.2 24.4 21.4 21.8

20.8 23.1 22.5 22.5 24.3 25.1 23.9 25.4 26.7

27.4 24.0 23.6 23.1 21.7 24.5 23.7 24.1 24.26

27.0 24.2 23.7 25.0 22.9 22.5 24.5 24.8 23.7

20.42 22.87 22.40 21.99 23.47 24.50 23.07 24.78 25.13

23.5 22.8 21.3 25.7 21.9 22.0 24.0 23.8 24.5

24.2 21.6 22.9 25.6 20.7 21.6 23.9 22.9 22.7

Solvent greatly influences the scavenging behavior of antioxidant compounds. The flavonoids scavenge the oxidant through either hydrogen atom transfer or single electron transfer. Structure of flavonoids and their complexes, their stoichiometry, solubility and solvent system are factors determining the scavenging mechanism [28]. Provision of hydrogen bonding in antioxidant makes them sensitive to solvent effects. This leads us to study solvent dependence of RSA, as a clear evidence of involvement of solute-solvent interaction and hydrogen bonding in hydrogen donation by antioxidant [29,30]. Hydrogen bonding acceptor of solvent molecule generally decreases the antioxidant activity of phenolic hydroxyl groups through intermolecular hydrogen bonding. Therefore, in solvent mixtures the antioxidant property of the species would decline with the increase of nonpolar component. In that case linear dependence of antioxidant activity

Table 6 Bond dissociation enthalpy in kcal/mol for Fls & M-Fls. Bond Dissociation Enthalpy (Kcal/mol) Fl

–OH at site

Free Fl

Cu-Fl

Fe-Fl

prim mo

Ro R1 R2 R3 R4 R5 R1 R2 R3 R4 R5

76.9 89.5 87.7 91.2 88.8 82.3 78.3 80.0 79.2 81.6 74.7

– 74.7 74.3 76.9 72.1 68.7 70.1 70.3 73.3 74.4 66.6

– 72.0 73.5 75.8 71.7 65.5 69.6 69.2 69.5 68.5 62.5

quer

on some empirical parameters would reveal some valuable information [31]. One of several empirical ways to analyze the effects of solvent in organic-water binary mixtures is to evaluate RSA in aqueous media. Fig. 5 reveals that the RSA values of all the compounds (the flavonoids and their metal chelates) increase with increasing solvent polarity parameter. The increase in RSA value can be attributed to increase in transferring of a hydrogen atom from the flavonoids to free radical caused by a solvent system with higher polarity and hydrogen bond donating ability. The RSA values of the flavonoids as well as their Cu2+ and

3.9

3.6

log[H2O] +Log (RSA)

3.5. Solvent effect on RSA of DPPH•

Fe-prim

Cu-prim

Fe-mo

Cu-mo

Fe-quer

Cu-quer

prim

quer

mo

3.3

3

2.7

2.4 0.0125

0.0145

0.0165

0.0185

1/ ε Fig. 5. The Yasuda-Shedlovsky plots of the metal-flavonoid complexes in different aqueous solutions of methanol.

438

E. Jabeen et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 171 (2017) 432–438 Total ROS units

4. Conclusions

SOD activity (units/min)

Cu2+ and Fe3+ complexes of three flavonoids morin (mo), quercetin (quer) and 5-hydroxyflavone (prim) were synthesized and dissolved in methanol-water mixtures. The scavenging activity as studied using UV– Vis spectroscopy and cyclic voltammetry under imitated physiological conditions revealed metal-flavonoid complexes exhibit better antioxidant activity than pure ligands. DFT calculations also predicted the same trends. Linear dependence of antioxidant activity on solvent polarity makes reliable extrapolation to zero organic solvent possible. Metal ions have an impact on the hydrogen atom transferring ability of the complex revealing that complexes deactivate oxidants through hydrogen atom transfer. Fe3 +-prim expressed itself as antioxidant through direct scavenging of free radicals as well as possesses boosting potential through increasing CAT and SOD activities endogenously.

400 300 200

CAT activity (units/min)

100 Total ROS units SOD activity (units/min)

0 control

Fe-prim

CAT activity (units/min)

alloxan

alloxan+ Fe-prim * SOD activity is reported here as 50 times of orgional value for comparative purpose Fig. 6. Graphical representation of total ROS units, SOD activity and CAT activity analysis of control, Fe3+-prim injected, alloxan treated and alloxan + Fe3+-prim treated compounds.

Fe3+ complexes within pure water were then determined by extrapolation of Yasuda–Shedlovsky approach in different methanol-water mixtures. This approach is usually applied for study of weak acids or bases in methanol-water mixture [32]. However, in this work we related empirical dielectric constant with RSA to evaluate the RSA values of the flavonoids and their complexes in pure water. The RSA of metal flavonoid complexes is in order of:

Acknowledgments We acknowledge Quaid-i-Azam University Islamabad for the provision of funds as URF and HEC Pakistan for funding through Project No. 20-1718/R&D/2011.

Appendix A. Supplementary data Cu2 +-quer N Fe3 +-quer N Cu2 +-mo N Fe3 +-mo N Fe3 +-prim NCu2 +prim It is interesting to analyze the result that trend of Cu2 + and Fe3 + complexes of flavonoids in methanol-water mixture gets revert in pure water except for mo complex Fig. 4. This reveals that solvent not only affects properties of individual complex but also controls the relative antioxidant activity. 3.6. In-vivo antioxidant activity of Fe3+-prim For biological activity assay, in vivo trials were made with one of the synthesized metal complex Fe3+-prim. The results of serum analysis concerning total ROS units, CAT activity and SOD activity are displayed in Fig. 6 where the horizontal rows express the result of given type of analysis while the columns represent the group treatment of rats. Alloxan is known to cause the diabetes in laboratory animals through damaging beta cells in liver [33]. During diabetic condition ROS level significantly increases in the body due to inadequate body defense system including antioxidants enzymes such as SOD and CAT. These enzymes play a key role in the scavenging of free radicals. Compound that can suppress the oxidative stress through either scavenge the free radicals or via up-regulation of CAT or SOD activities, would be regarded as antioxidant [34]. It is clear from the Fig. 5 that the alloxan treated group display high ROS units and lower SOD and CAT activities as compare to control group which has only been treated with normal saline. Considering the compounds effect over rats; (compare ‘alloxan with alloxan + compound’ and compare ‘compound with control’), it is clear that injection of compound has resulted in decrease in ROS units and elevation of ROS and SOD activities in both alloxan pretreated and normal groups. CAT and SOD activities of the compound injected group is slight is less under the condition of alloxan pretreatment than normal pretreatment, this is because the alloxan has resulted in significant suppression of SOD and CAT already which has to be restored by the Fe3+-prim. Thus, Fe3+-prim has displayed itself as antioxidant in in-vivo system by decreasing total ROS (units) and at the same time through enhancement of SOD and CAT activities.

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.saa.2016.08.035.

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