A study on the antioxidant activity of pyridylselenium compounds and their slow release from poly(acrylamide) hydrogels

Article abstract of DOI:10.1080/10426507.2013.844143

The antioxidant activity of pyridylselenium compounds has been evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical and nitric oxide (NO) scavenging methods. Pyridylselenium compounds have shown far superior (100-1000 times) antioxidant property than ebselen. The control release of bis(2-pyridyl) diselenide from poly(acrylamide) hydrogels has been studied in order to evaluate its release mechanism and diffusion coefficient. The later study also demonstrates that the pyridylselenium loading into the polymer matrix increases the magnitude and the rate of the radical scavenging activity of the poly(acrylamide) hydrogels. Copyright

Full text of DOI:10.1080/10426507.2013.844143

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Phosphorus, Sulfur, and Silicon and the
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A Study on the Antioxidant Activity of
Pyridylselenium Compounds and their
Slow Release from Poly(acrylamide)
Hydrogels
Jaspreet S. Dhaua, Avtar Singha, Amritpal Singha & Balwinder S.
Soochb
a Department of Chemistry, Punjabi University, Patiala-147002,
Punjab, India
Click for updates
b Department of Biotechnology, Punjabi University, Patiala-147002,
Punjab, India
Accepted author version posted online: 30 Sep 2013.Published
online: 14 May 2014.
To cite this article: Jaspreet S. Dhau, Avtar Singh, Amritpal Singh & Balwinder S. Sooch (2014)
A Study on the Antioxidant Activity of Pyridylselenium Compounds and their Slow Release from
Poly(acrylamide) Hydrogels, Phosphorus, Sulfur, and Silicon and the Related Elements, 189:5, 687-699,
DOI: 10.1080/10426507.2013.844143
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Phosphorus, Sulfur, and Silicon, 189:687–699, 2014
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2013.844143
A STUDY ON THE ANTIOXIDANT ACTIVITY OF
PYRIDYLSELENIUM COMPOUNDS AND THEIR SLOW
RELEASE FROM POLY(ACRYLAMIDE) HYDROGELS
Jaspreet S. Dhau,1 Avtar Singh,1 Amritpal Singh,1
and Balwinder S. Sooch2
1Department of Chemistry, Punjabi University, Patiala-147002, Punjab, India
2Department of Biotechnology, Punjabi University, Patiala-147002, Punjab, India
GRAPHICAL ABSTRACT
Abstract The antioxidant activity of pyridylselenium compounds has been evaluated by 1,1-
diphenyl-2-picrylhydrazyl (DPPH) free radical and nitric oxide (NO) scavenging methods.
Pyridylselenium compounds have shown far superior (100–1000 times) antioxidant property
than ebselen. The control release of bis(2-pyridyl) diselenide from poly(acrylamide) hydrogels
has been studied in order to evaluate its release mechanism and diffusion coefficient. The later
study also demonstrates that the pyridylselenium loading into the polymer matrix increases the
magnitude and the rate of the radical scavenging activity of the poly(acrylamide) hydrogels.
Keywords Pyridylselenium; Antioxidant; Control release; Poly(acrylamide) hydrogels
INTRODUCTION
Organoselenium compounds have attracted considerable attention worldwide be-
cause of their broad spectrum of biological activities.1–9 These compounds have shown
Received 10 June 2013; accepted 8 September 2013.
We are thankful to the CSIR, New Delhi, India, for financial support. Additional support from SAP by UGC,
New Delhi, is also acknowledged.
Address correspondence to Jaspreet S. Dhau, Department of Chemistry, Punjabi University, Patiala-147002,
Punjab, India. E-mail: jassiv02@yahoo.co.in
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/gpss.
687

688
J. S. DHAU ET AL.
Figure 1 Pyridylselenium compounds.
tremendous scope as antioxidant, enzyme inhibitor, cytokine inducer, immunomodu-
lator, and antimicrobial agents.10–14 Several reports have been published on the glu-
tathione peroxidase (GPX) mimetic activity of the selenium compounds.15–20 Ebselen (2-
phenyl-1,2-benzisoselenazole-3 (2H)-one), a nontoxic mimetic of GPX, has demonstrated
protection against brain ischemia and stroke.21,22 The low molecular weight diorganyl
diselenides have shown pharmacological effects such as antioxidant, hepatoprotective,
neuroprotective, anti-inflammatory, and antinociceptive properties.23–29 In light of the fact
that the replacement of an aromatic ring with a pyridine moiety boost the biological
activity of the organylselenium compounds,30,31 the present work is an endeavor to eval-
uate the antioxidant activity of various methyl-substituted pyridylselenium compounds
(Figure 1) by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals and NO scavenging
methods.
In addition to its potential beneficial properties, the organoselenium compounds have
exhibited cytotoxicity and genotoxicity at higher concentrations.32,33 These compounds
generate free radicals and break DNA strand at elevated concentrations.32,33 It is clear from
the later discussion that only a narrow therapeutic window exists for the organoselenium
compounds and the conventional drug delivery systems would not provide ideal pharma-
cokinetic profiles for these compounds. The ideal pharmacokinetic profile can be attained
by reaching the therapeutic levels without exceeding the maximum endurable dose while
maintaining the required concentration. The later objective could be achieved by the use
of a polymer matrix as a drug-releasing device which releases organoselenium compounds
in a controlled and sustained manner. We herein report for the first time, a study on the
slow release of the titled compounds from the poly(acrylamide) hydrogels (acrylamide =
AAm).
RESULTS AND DISCUSSION
Antioxidant Activity Studies
The antioxidant property of the pyridylselenium compounds has been evaluated by
the DPPH free radicals34 and NO scavenging methods.35 There are relatively few studies
on the antioxidant property of the organoselenium compounds by these two methods. This
is probably due to the fact that ebselen and its substituted derivatives have shown little
promise as a radical scavenger against DPPH radicals.36

A STUDY ON THE ANTIOXIDANT ACTIVITY OF PYRIDYLSELENIUM COMPOUNDS 689
Table 1 DPPH radical percent scavenging activity of pyridylselenium compounds
Percent scavenging
activity (%)
Percent scavenging
activity (%)
Compound
Conc. (μM)
1.0 × 105
Compound
Conc. (μM)
2.0 × 102
Bis(2-pyridyl)
diselenide (1a)
30.70 1.34
2-Pyridylselenenyl
bromide (2a)
38.59 1.42
5.0 × 104
2.5 × 104
1.0 × 105
20.95 2.15
10.19 1.05
57.96 1.69
1.0 × 102
0.5 × 102
2.0 × 102
27.74 1.62
13.33 1.52
65.01 1.03
Bis(3-methyl-2-
pyridyl)
diselenide (1b)
3-Methyl-2-pyridyl
selenenylbromide
(2b)
5.0 × 104
2.5 × 104
1.0 × 105
45.07 0.99
30.40 1.52
44.82 1.12
1.0 × 102
0.5 × 102
2.0 × 102
61.86 1.21
56.92 1.67
57.71 1.54
Bis(4-methyl-2-
pyridyl)
diselenide (1c)
4-Methyl-2-pyridyl
selenenylbromide
(2c)
5.0 × 104
2.5 × 104
1.0 × 105
36.15 1.14
17.25 0.65
33.83 1.25
1.0 × 102
0.5 × 102
2.0 × 102
54.02 0.81
48.55 0.83
38.63 1.42
Bis(5-methyl-2-
pyridyl)
diselenide (1d)
5-Methyl-2-pyridyl
selenenylbromide
(2d)
5.0 × 104
2.5 × 104
1.0 × 105
20.00 1.02
11.80 0.67
52.11 1.01
1.0 × 102
0.5 × 102
2.0 × 102
31.36 1.08
28.40 1.21
72.07 0.89
Bis(6-methyl-2-
pyridyl)
diselenide (1e)
6-Methyl-2-pyridyl
selenenylbromide
(2e)
5.0 × 104
2.5 × 104
5.0 × 104
41.89 1.70
30.00 1.02
69.96 0.94
1.0 × 102
0.5 × 102
2.0 × 102
68.18 0.57
54.26 0.27
77.19 0.16
Bis(3,5-dimethyl-
2-pyridyl)
3,5-Dimethyl-2-
pyridyl
diselenide (1f)
selenenylbromide
(2f)
2.5 × 104
5.0 × 104
51.40 1.23
81.00 1.00
1.0 × 102
0.5 × 102
5.0 × 104
70.88 0.53
66.93 1.00
31.95 2.48
Bis(4,5-dimethyl-
2-pyridyl)
Ebselen
diselenide (1g)
2.5 × 104
72.10 1.15
2.5 × 104
17.66 1.47
DPPH Radical Scavenging Method
The data from the DPPH radical scavenging method reveal increase in the antioxidant
activity of the pyridylselenium compounds with the increase in the number of methyl groups
attached to the pyridine ring (One-way ANOVA, p < 0.05 by Turkey test; Table 1). Among
the diselenides, bis(2-pyridyl) diselenide (1a) shows the minimum and bis(3,4-dimethyl-2-
pyridyl) diselenide (1f) exhibits the maximum antioxidant activity (Table 1 and Figure 2a).
In case of pyridylselenenylbromides, 2f shows the maximum and 2a displays the mini-
mum antioxidant activity (Table 1 and Figure 2b). 2-Pyridylselenenylbromides have been
found to be approximately 1000 times more active than the parent diselenide and ebselen
(Table 1).
Analysis of the data obtained with picolyselenium compounds (1b–1e and 2b–2e)
suggests decrease in the antioxidant activity with the increase in the bond distance between
the selenium moiety and the methyl group (Figure 3, the bond distances were calculated
from the crystal structure of the respective diselenides37,38).

690
J. S. DHAU ET AL.
90
80
70
60
50
40
30
20
10
0
80
70
60
50
40
30
20
10
0
1a
1b
1c
1d
1e
1f
1g ebselen
2a
2b
2c
2d
2e
2f
Compound
Compound
a
b
Figure 2 Comparison of DPPH scavenging activity: (a) diselenides at 5 × 104 μM and (b) 2-pyridylselenenyl
bromides at 100 μM. Data are reported as mean SD of three readings per concentration.
(1b/2b)ortho-methyl(C-3) > (1e/2e)meta-methyl(C-6) > (1c/2c)meta-methyl(C-4)
> (1d/2d)para-methyl(C-5).
Two-way ANOVA of DPPH activity yielded a significant Se–CH3 distance (Å) × con-
centration of pyridylselenium interaction (p < 0.05 by Turkey test). This result indicates sig-
nificant role of the electron-releasing inductive effect of the methyl group in enhancing the
antioxidant activity of the titled compounds. Interestingly, 2,6-bis(3,5-dimethylselenenyl)
pyridine (1h), which contains two selenium atoms at two different positions and no Se–Se
bond, does not show the DPPH radical scavenging activity at any concentration. From
50
40
30
20
10
0
4.842(1b)
6.07(1e)
6.193(1c)
7.455(1d)
Distance between selenium and methyl group(compound)
Figure 3 Relation between Se–CH3 distance (Å) and DPPH reduction activity of diselenides (1b–1e). The bond
distances were calculated from the crystal structure of the respective diselenides.37,38 Results are reported as
mean SD of three readings per concentration.

A STUDY ON THE ANTIOXIDANT ACTIVITY OF PYRIDYLSELENIUM COMPOUNDS 691
70
80
60
50
40
30
20
10
0
70
60
50
40
30
20
10
0
1a
1b
1c
1d
1e
1f
1g ebselen
2a
2b
2c
2d
2e
2f
Compound
Compound
a
b
Figure 4 Comparison of NO percent scavenging activity: (a) diselenides at 400 μM and (b) 2-
pyridylselenenylbromides at 2 × 104 μM.
these observations, it could be inferred that the presence of Se X (X = Sepy, Br) bond is
essential for the antioxidant activity. It also appears that the antioxidant quenching cycle
involves the generation of free radicals (pySe· and X·) due to the cleavage of the Se–Br
and Se Se bond induced by the DPPH radical in the solution. It is postulated that the
electron-releasing methyl group stabilizes the pySe· radical which drives the cleavage of
the Se X bond. The lower bond dissociation energy for the Se Br (297.0 kJ/mole) bond
than for the Se–Se (332.6 kJ/mole) bond39 partly explains the greater antioxidant activity
of the selenenylbromides than the corresponding diselenides.
Table 2 NO scavenging activity of pyridylselenium compounds
Percent scavenging
activity (%)
Percent scavenging
activity (%)
Compound
Conc. (μM)
Compound
Conc. (μM)
1a
4.0 × 102
2.0 × 102
1.0 × 102
4.0 × 102
2.0 × 102
1.0 × 102
4.0 × 102
2.0 × 102
1.0 × 102
4.0 × 102
2.0 × 102
1.0 × 102
4.0 × 102
2.0 × 102
1.0 × 102
4.0 × 102
2.0 × 102
1.0 × 102
4.0 × 102
2.0 × 102
1.0 × 102
40.55 1.27
30.01 0.89
17.86 1.04
56.10 1.11
42.53 1.30
34.31 0.85
47.43 0.72
37.17 1.45
48.55 1.55
44.65 0.96
32.47 1.24
27.25 1.69
54.66 0.74
45.21 0.82
30.70 1.01
70.49 0.94
57.70 0.74
35.47 1.46
77.30 0.64
62.61 1.10
49.41 1.00
2a
2.0 × 104
1.0 × 104
5.0 × 103
2.0 × 104
1.0 × 104
5.0 × 103
2.0 × 104
1.0 × 104
5.0 × 103
2.0 × 104
1.0 × 104
5.0 × 103
2.0 × 104
1.0 × 104
5.0 × 103
2.0 × 104
1.0 × 104
5.0 × 103
8.0 × 102
6.0 × 102
4.0 × 102
29.66 1.52
21.22 1.06
13.69 1.84
53.22 0.99
43.40 0.58
33.59 1.19
49.04 0.97
37.69 1.03
48.55 0.91
43.38 1.25
32.09 0.76
22.88 1.83
52.17 1.48
42.18 1.41
26.76 1.12
62.82 1.16
52.55 1.38
41.67 0.60
52.16 0.86
43.90 1.44
32.30 1.13
1b
1c
1d
1e
1f
2b
2c
2d
2e
2f
1g
Ebselen

692
J. S. DHAU ET AL.
Table 3 DPPH radical scavenging activity of 2b-loaded and unloaded poly(AAm) hydrogels
Percent scavenging activity
of poly(AAm) hydrogel (%)
Percent scavenging activity
of 2b-loaded poly(AAm) hydrogel (%)
S. No.
Time (min)
1.
2.
3.
4.
5.
6.
7.
8.
45
90
2.30
3.20
4.60
7.30
9.50
10.68
11.07
11.47
2.86
4.83
5.09
135
180
225
270
315
360
8.05
12.28
13.48
15.00
15.84
NO Scavenging Method
Contrary to the results obtained with the DPPH radical reduction method, the dis-
elenides show greater NO scavenging activity than the corresponding pyridylselenenyl-
bromides (one-way ANOVA/Turkey, p < 0.05). In fact, the diselenides are approximately
100 times more active than the pyridylselenenylbromides. This indicate different modes of
action of organoselenium compounds in the quenching of NO and DPPH radicals. Impor-
tantly, all the diselenides shows greater (1.5–2.0 times) NO antioxidant activity than ebselen
(Table 2 and Figure 4a). The NO scavenging efficacy also increases with the increase in the
number of methyl groups attached to the pyridine ring (Figures 4a and 4b).
Antioxidant Activity of Unloaded and Selenium-Loaded
Poly(AAm) Hydrogels
The antioxidant activity of poly(AAm) hydrogels was determined by the DPPH rad-
ical scavenging method. The activity was determined after fixed interval of time (Table 3).
The maximum antioxidant activity of the hydrogel was observed after 9 h of incubation at
room temperature. The antioxidant activity of the polymer could be due to partial quenching
of the DPPH radical by the pendant free radical groups in the polymer matrix (Figure 5).
Also, the diffusion of DPPH radicals into the polymer matrix (sorption of DPPH into the
polymer matrix) may be contributing to the reduction in the DPPH concentration in the
surrounding solution (Figure 5). Next, the antioxidant property of poly(AAm) hydrogels
loaded with 3-methyl-2-pyridylselenenylbromide (2b) was determined. The loading of 2b
Active pendant free radical groups
DPPH
DPPH solution
in methanol
Poly(AAm) hydrogel matrix
Beaker
Poly (AAm) hydrogel Cross-section (top view)
DPPH solution in methanol
Figure 5 Schematic of the mode of antioxidant activity of poly(AAm) hydrogels.

A STUDY ON THE ANTIOXIDANT ACTIVITY OF PYRIDYLSELENIUM COMPOUNDS 693
non-loaded
loaded
16
14
12
10
8
6
4
2
0
50
100
150
200
250
300
350
400
Time(min)
Figure 6 DPPH radical scavenging activity of 2b-loaded and nonloaded poly(AAm) hydrogels.
into poly(AAm) hydrogels has been discussed later in Experimental section. It is clearly
visible from Figure 6 that the loading of the compound, 2b, into the polymer matrix in-
creases the magnitude and the rate of radical scavenging activity of poly(AAm) hydrogels.
This may be due to the slow release of 2b from the polymer matrix into the surrounding
solution where it quenches the DPPH radicals (Figure 7). It is also possible that some of
the DPPH radicals diffuse into the polymer matrix, where they react with the trapped 2b
molecules (Figure 7). The effect of bis(2-pyridyl) diselenide loading on the antioxidant
activity of the hydrogel was also studied. However, no significant increase in the activity of
the hydrogels was noticed. This is due to the lower activity of the diselenides in comparison
to the corresponding pyridylselenenylbromides.
Release Dynamics of 1a from the Loaded Poly(AAm) Hydrogels
in Different Medium
The release profile of 1a from the loaded polymer matrix in different release medium
at 37◦C is presented in Figure 8. The amount of 1a released in pH 2.2 buffer was higher
than the release in pH 7.4 buffer solution. After 24 h, the total amount of 1a released in pH
2.2 buffer and pH 7.4 buffer was found to be 2.869 0.110 and 2.063 0.184 mg/10 mL/g
of hydrogel, respectively. The greater release of 1a in pH 2.2 buffer may be due to its higher
solubility in the acidic conditions. From Figure 8, it is clear that release of 1a from the drug
loaded polymer occurred in a controlled manner. The values of diffusion exponent “n” and
gel characteristic constant “k” for the release of 1a from drug loaded hydrogels in different
releasing medium was evaluated from the slope and intercept of the plot of ln Mt/M versus
∞

694
J. S. DHAU ET AL.
Poly(AAm) hydrogel
2b solution in methanol (24 hours)
2b-loaded poly(AAm) hydrogel
DPPH solution in methanol
Poly(AAm)
hydrogel matrix
3-MePySeBr
DPPH
DPPH-(3-MePySeBr)
Beaker
DPPH-3-MePySeBr Solution
Figure 7 Schematic of the mode of antioxidant activity of 2b-loaded poly(AAm) hydrogels.
2.2 pH buffer
7.4 pH buffer
3.0
2.5
2.0
1.5
1.0
0.5
0
60
120
180
240
300
1440
Time (min.)
Figure 8 Release profile of 1a from loaded poly(AAm) hydrogels in different release medium at 37◦C.

A STUDY ON THE ANTIOXIDANT ACTIVITY OF PYRIDYLSELENIUM COMPOUNDS 695
ln t. The values of “n” and “k” at pH 2.2 buffer is determined to be 0.629 and 23.841 ×
10−3, respectively. At pH 7.4 buffer, these values are found to be 0.521 and 33.102 × 10−3
,
respectively. The release of 1a in pH 2.2 and pH 7.4 buffers occurred through non-Fickian
diffusion mechanism.40,41 In this mechanism, the rate of diffusion of water molecules into
the polymer matrix and the rate of polymer chains relaxation are comparable.
CONCLUSION
In summary, we have demonstrated that the presence of electron releasing methyl
group and its position on the pyridine ring influences the antioxidant activity of the
pyridylselenium compounds. The slow release study shows the potential of poly(AAm)
hydrogels as the organylselenium-release device for controlling the selenium related toxic
effects. It has also been demonstrated that the pyridylselenium loading in poly(AAm)
hydrogels increases the magnitude and the rate of the antioxidant activity of the
hydrogels.
EXPERIMENTAL
Materials and General Characterization
Acrylamide (AAm), N,N,Nꢁ,Nꢁ-tetramethylethylenediamine (TMED) and
DPPH were obtained from Merck Specialities Private Limited Mumbai-India, N,N’-
methylenebisacrylamide (NN-MBA) and ammonium persulfate (APS) were obtained
from Qualigens Fine Chemicals Mumbai-India. Bis(2-pyridyl)-, bis(3-methyl-2-pyridyl)-
and bis(6-methyl-2-pyridyl) diselenides were prepared by literature method involving
elemental selenium, hydrazine hydrate and the respective 2-bromopyridines.37 The
diselenides of 4- and 5-picoline, 3,5-, and 3,4-lutidine were synthesized by the lithiation of
picolinium-BF3 or lutidinium-BF3 adduct with lithium diisopropylamide (LDA) at –78◦C
followed by selenium insertion reaction and aerial oxidation.42,43 The later methodology
was also used to synthesize 3,5-dimethyl-2,6-bis(methylselenenyl)pyridine (1h).42 Further,
all the synthesized diselenides were reacted with bromine in dichloromethane to afford
the corresponding 2-pyridylselenenylbromides in near quantitative yields.38 1H NMR and
13C NMR spectra were recorded on Brucker 400 MHz spectrophotometer in CDCl3 using
tetramethylsilane (TMS) as an internal standard. EI mass spectra were taken by using
GC–Mass spectrometer [GCMS QP-2010 plus] with Rtx-1MS (30 m × 0.25 mm i.d. ×
0.25 μm) capillary column. The spectroscopic data of all the synthesized compounds
agreed well with the literature values.37,38,42,43 The UV–Vis spectrophotometer experiments
were carried on Shimadzu PharmaSpec UV-1700, spectrophotometer.
Antioxidant Activity Assay
Reduction of DPPH Free Radical Method. The method given by Krings et al.
was used for this assay34 with slight modifications. The solutions of different concentration
of test compounds were added to DPPH (250 μM) in methanol. The testtubes were kept at
room temperature for 45 min and the absorbance was measured at λ 517 nm. The difference
between the test and the controlled experiments was taken and expressed as the percentage
scavenging activity. The percentage scavenging activity was calculated according to the

696
J. S. DHAU ET AL.
following formula:
% Scavenging activity = [control − test]/control × 100
Assay for Nitric Oxide (NO) Scavenging Activity
The method of Belsare et al. was used for this assay.35 Sodium nitroprusside (5 mM)
in phosphate buffer pH 7.4 was incubated with different concentrations of test compounds
dissolved in methanol. The testtubes containing the test compounds were incubated at 25◦C
for 2 h. The control experiment was conducted with equal amount of solvent in identical
manner. After incubation, 2 mL of incubated solution was taken and diluted with 2 mL of
Griess reagent (1% sulfanilamide, 0.1% N-naphthylethylenediamine dihydrochloride and
2% o-phosphoric acid dissolved in distilled water). Absorbance of the solution was read at
λ 546 nm. The percentage scavenging activity was calculated according to the following
formula:
% Scavenging activity = [control − test]/control × 100
Preparation of Poly(AAm) Hydrogels
Reaction was carried out with 70.34 × 10−2 mol/L of AAm, definite concentration
of initiator APS (13.15 × 10−3 mol/L) and crosslinker NN-MBA (32.43 × 10−3 mol/L),
taken in the aqueous reaction system in the presence of accelerator TMED (100 μL) at
room temperature for 2 h. The crosslinked hydrogel was then stirred in distilled water
to remove the soluble fractions and then were dried in oven at 40◦C. The crosslinked
polymers were named as of poly(AAm) hydrogels. These polymers are used to study
the effect of pH on the release dynamics of the drug (pyridylselenium compound) from
the hydrogels for the evaluation of mechanism of drug release and various diffusion
coefficients.
Antioxidant Activity of Unloaded and Selenium-Loaded
Poly(AAm) Hydrogels
100 mM solution of 3-methyl-2-pyridylselenenylbromide (2b) was loaded in
poly(AAm) hydrogels by dissolving it in a heterogeneous mixture containing 0.1 g of
the hydrogel soaked in methanol for 24 h. The 2b-loaded hydrogel was taken out from
the mixture and air dried. The surface of the dried material was washed with methanol
to remove 2b from the surface. This gives poly(AAm) hydrogel with trapped 2b in the
polymer matrix. The washed and dried loaded-hydrogel was taken in a testtube and a
solution of DPPH (250 μM) in methanol was added to it. In another testtube, a solution of
DPPH (250 μM) in methanol and 0.1 g of poly(AAm) hydrogel was taken. In both the test
mixtures, the total volume of solution was kept at 4 mL. The control in this setup contains
the DPPH solution in methanol. The testtubes were kept at room temperature for 1 h and
the absorbance was measured at λ 517 nm. Absorbance of test and control were recorded
after a fixed interval of time.

A STUDY ON THE ANTIOXIDANT ACTIVITY OF PYRIDYLSELENIUM COMPOUNDS 697
Release Dynamics of bis(2-pyridyl) Diselenide (1a) from the Loaded
Poly(AAm) Hydrogels
The release profile of 1a from the loaded polymer matrix was determined in pH
2.2 buffer and pH 7.4 buffer. The pH of the release medium was selected in order to
simulate the release profile of the 1a in acidic condition in stomach (pH 1–2) and neutral
condition in intestine (pH 7.4 in terminal ileum).44 All the studies were carried out in
triplicate. Preparation of buffer solution, calibration curves, drug loading, drug release,
and preparation of reagents have been performed according to the literature method.41 The
calibration curves of 1a were prepared in 2% HCl solution, pH 2.2 buffer and pH 7.4
buffer solution at λmax 279 nm, 277 nm, and 281 nm, respectively. The loading of 1a into
poly(AAm) hydrogel was carried out by swelling an equilibrium method. The poly(AAm)
hydrogel was allowed to swell in a solution of known concentration of 1a (400 μg/mL)
for 24 h at 37◦C and then dried to obtain the release device. Entrapment efficiency (%) of
poly(AAm) hydrogel was determined to be 28.48 0.17%. In vitro release studies of 1a
were carried out by placing dried and 1a loaded sample in a definite volume (10 mL) of
releasing medium for 30 min at 37◦C and then sample was transferred to the fresh releasing
medium. The release of 1a from the polymer samples was measured from the calibration
graphs which were prepared on the UV–VIS spectrophotometer. The mechanism of 1a
release was determined by using equation given by Ritger and Peppas40
Mt
= ktn,
M
∞
where Mt/M is the fractional release of drug in time t, “k” is the constant characteristic
∞
of the drug–polymer system, and “n” is the diffusion exponent characteristic of the release
mechanism. Mt and M are drug released at time “t” and at equilibrium, respectively.40
∞
Statistical Analysis
The results are presented as means SD. Statistical analysis was performed using
a one-way and two-way analysis of variance (ANOVA), followed by Turkey test. P values
less than 0.05 (P < 0.05) were used as an indicator of significance.
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