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Syntheses and Biological Activities of Benzimidazolo[21-b] benzo[e]thiazepin-510H-ones.

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Arch. Pharm. Chem. Life Sci. 2008, 341, 49 – 54
N. Rastkari et al.
49
Full Paper
Syntheses and Biological Activities of Benzimidazolo[2,1-b]
benzo[e]thiazepin-5(10H)-ones
Noushin Rastkari1, 3, Mohammad Abdollahi2, Reza Ahmadkhaniha3, and Abbas Shafiee3
1
Center for Environmental Research, Tehran University of Medical Sciences, Tehran, Iran
Department of Toxicology and Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences Research
Center, Tehran University of Medical Sciences, Tehran, Iran
3
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center,
Tehran University of Medical Sciences, Tehran, Iran
2
Substituted benzimidazolo[2,1-b]benzo[e]thiazepin-5(10H)-one derivatives were prepared in moderate to good yield by reaction of mercapto benzimidazole derivatives with 2-chloromethylbenzoyl chloride as a coupling component. Their structures were confirmed by elemental analysis,
IR, NMR, and MS. Their antioxidant properties were evaluated by several methods: scavenging
effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, reducing power assay, and inhibition of
lipid peroxidation. In DPPH assay, 3h exhibited an activity stronger than trolox. In the reducing
power assay and inhibition of lipid peroxidation, compound 3a was more active than 3h and
similar to trolox. The antidiabetic effect was also determined, and the antidiabetic activities
were correlated with their antioxidant properties, compound 3a was the most active compound
in the in-vivo experiments.
Keywords: Antioxidant activity / Benzothiazepine / Synthesis /
Received: May 12, 2007; accepted: September 20, 2007
DOI 10.1002/ardp.200700099
Introduction
Benzothiazepine derivatives exhibit diverse biological
activities. They may act as calcium channel antagonists
[1], angiotensin-converting enzyme inhibitors [2], anticonvulsants, tranquilizers [3], or anticancer agents [4],
and possess endogenous natriuretic activities [5]. Though
it is known that Benzothiazepines exhibit different biological activities, it is the first time that a study on their
antioxidant properties is reported.
The search for new molecules with antioxidant properties is a very active domain of research, since recent evi-
Correspondence: Abbas Shafiee, Department of Medicinal Chemistry,
Faculty of Pharmacy and Pharmaceutical Sciences Research Center,
Tehran University of Medical Sciences, Tehran, 14174 Iran.
E-mail: ashafiee@ams.ac.ir
Fax: +98 21 664-61178
Abbreviations: 1-diphenyl-2-picrylhydrazyl (DPPH); radical scavenging
activity (RSA); ferric-reducing ability of plasma (FRAP); thiobarbituric acid
reactive substances (TBARS)
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2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
dence [6] suggests that free radicals, which are generated
in many bioorganic redox processes, may induce oxidative damage in various components of the body (e. g. lipids, proteins, and nucleic acids) and may also be involved
in processes leading to the formation of mutations. Furthermore, radical reactions play a significant role in the
development of life-limiting chronic diseases such as cancer, hypertension, cardiac infraction, arteriosclerosis,
rheumatism, cataracts, and others [7]. A key factor in the
induction of oxidative stress appears to be the overproduction of free radicals typically caused by avitaminosis
A, C, and E and reduced levels of specific enzymes such as
superoxide dismutase, catalase, and glutathione peroxidase. One important way to protect the body against
such stress is to increase the level of antioxidants [8].
Such compounds may play a significant role in the prevention or alleviation of the above-mentioned diseases by
reducing oxidative damage to cellular components
caused by reactive oxidant species [9 – 12].
As a part of our ongoing program to design novel antioxidant candidate [13, 14], herein, we report the synthe-
50
N. Rastkari et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 49 – 54
Table 1. Physical data of synthesized compounds 3a – h.
Compound
R1
R2
Mp. (8C)
Yield (%)
Recrystallization
solvent
Molecular
formula
3a
3b
3c
3d
3e
3f
3g
3h
H
H
H
H
H
CH3
NO2
H
H
CH3
NO2
Cl
Br
H
H
OH
175 – 177
168 – 170
210 – 212
178 – 180
234 – 237
162 – 164
201 – 203
140 – 142
65
70
58
65
70
38
60
40
Acetone
Acetone
Acetonitrile
Acetone
Acetone
Acetone
Acetonitrile
Acetonitrile
C15H10N2OS
C16H12N2OS
C15H9N3O3S
C15H9ClN2OS
C15H9BrN2OS
C16H12N2OS
C15H9N3O3S
C15H10N2O2S
sis and biological screening of a number of benzimidazolo[2,1-b]benzo[e]thiazepin-5(10H)-ones.
Results and discussion
Substituted
benzimidazolo[2,1-b]benzo[e]thiazepin5(10H)-one derivatives were prepared in moderate to
good yield (Table 1 and Scheme 1) and their antioxidant
properties were evaluated by several methods (scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, reducing power assay, and inhibition of lipid peroxidation). The antidiabetic effect was also determined.
Free radical scavenging is one of the best known mechanisms by which antioxidants inhibit lipid oxidation.
DPPH (1-diphenyl-2-picrylhydrazyl) radical scavenging
activity evaluation is a standard assay in antioxidant
activity studies and offers a rapid technique for screening
the radical scavenging activity (RSA) of specific compounds or extracts. DPPH is a stable, free radical that can
accept an electron or hydrogen radical and thus be converted into stable, diamagnetic molecule. DPPH has an
odd electron, and, therefore, has a strong absorption
band at 517 nm. When this electron becomes paired, the
absorption decreases with respect to the number of electrons taken up [15, 16]. The RSA of compounds 3a – h
were tested using a methanolic solution of the stable,
free radical, DPPH. A freshly prepared DPPH solution
exhibits a deep purple color with an absorption maximum at 517 nm. This purple color generally fades/disappears when an antioxidant is present in the medium.
Thus, antioxidant molecules can quench DPPH free radicals (i. e. by providing hydrogen atoms or by electron donation, conceivably via a free-radical attack on the DPPH
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2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Scheme 1. Synthesis route of compounds 1 – 3.
molecule) and convert them to a colorless product (i. e.
2,2-diphenyl-1-hydrazine or a hydrazine analogue),
resulting in a decrease in absorbance at 517 nm. Hence,
the more rapidly the absorbance decreases, the more
potent is the antioxidant activity of the compound. The
RSA values of methanolic solutions of compounds 3a – h
were examined and compared to trolox (Table 2). Results
are expressed as a percentage of the ratio of the decrease
in absorbance at 517 nm to the absorbance of DPPH solution in the absence of test compounds at 517 nm. From
analysis of Table 2, we can conclude that the test compounds' scavenging effects on DPPH radicals increase
with the concentration and were excellent for compounds 3a and 3h (85.2% and 93.3% at 0.25 g/L, respectively), similar or higher RSA values than the standard
trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxwww.archpharm.com
Arch. Pharm. Chem. Life Sci. 2008, 341, 49 – 54
Benzimidazolo[2,1-b]benzo[e]thiazepin-5(10H)-ones
51
Table 2. Scavenging activity (%) on DPPH radicals of compounds 3a – h.
Compound
3a
3b
3c
3d
3e
3f
3g
3h
Trolox
Concentration (g/L)
0.0156
0.0312
0.0625
0.125
0.25
40.2
10.5
26.6
19.9
15.9
3.1
5.9
55.8
39.5
60
15.6
33.8
25.4
22.4
7.5
10.5
85.8
61.1
73.9
18.4
42.7
30.1
25.5
9.8
15.5
88.4
74.5
83.1
24.1
53.4
39.8
32.2
12.8
19.7
91.1
83.8
85.2
33.3
67.8
45.2
41.4
15.5
29.2
93.3
85.6
Figure 2. Effect of compounds 3a – h and trolox on rat blood lipid
peroxidation. (a) the difference between control and treated
groups is significant at P a 0.05; (b) compound 3a and trolox had
similar activity.
Figure 1. Effect of compounds 3a – h and trolox on rat blood
antioxidant power. (a) the difference between control and treated
groups is significant at P a 0.05; (b) compound 3a and trolox had
similar activity.
ylic acid) (85.6%) at the same concentration. The RSA drastically decreased to 33.3 at 0.25 g/L for compound 3b
bearing a methyl group at the R2 position. Compounds
with a substituent at the R1 position, 3f and 3g, showed
very low RSA values.
Figure 1 shows the reducing power of a plasma sample
of animals which were treated with compounds 3a – h.
The antioxidant power of the plasma samples was determined by measuring their ability to reduce Fe3+ to Fe2+,
established as FRAP test, and was described previously by
Benzie and Strain [17]. The FRAP (ferric-reducing ability
of plasma) assay measures the ferric-reducing ability of
plasma or serum. The order of reducing power activity
was 3a A 3h A 3c A 3d A 3e A 3b > 3g A 3f. It is worthwhile
to mention that the presence of a substituent in the R2
position was much better than a substituent in the R1
position (3b and 3c in comparison with 3f and 3g, respectively). Consequently, compounds 3b and 3c have higher
reducing-power values. Compound 3a showed higher
reducing-power activity than 3h. Therefore, from the
results it could be concluded that compound 3h was
probably metabolized faster than compound 3a in vivo
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2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
because of its hydroxyl group. However, 3a had similar
activity to trolox.
To investigate the antioxidant activity, compounds
3a – h were evaluated for the inhibition of lipid peroxidation (Fig. 2). Malondialdehyde (MDA) and other end products of the oxidation of polyunsaturated fatty acids and
their concentrations in the medium, is an established
measure of lipid peroxidation [18, 19]. In this test the
reaction of lipid peroxides with thiobarbituric acid (TBA)
makes a complex that is determined spectrophotometrically and lipid peroxidation in samples are assessed in
terms of thiobarbituric acid reactive substances (TBARS)
produced. The calibration curve of a 1,1,3,3-tetrahydroxypropan standard solution was used to determine the concentrations of TBARS in samples. The results from this
experiment also confirmed that compounds having substitution at R1 (compounds 3f and 3g) were inactive. However, compounds having R2 substitution were more
active than the control; the most active compound was
3a which had activity similar to trolox.
Free radicals have been implicated as the cause of several disorders, which includes diabetes, and the agents
that scavenge free radicals may have a great potential in
ameliorating these disease processes. Antioxidants play
an important role in protecting the human body against
damage by reactive oxygen species. Increased oxidative
stress has been postulated in the diabetic state. Oxidative
stress in diabetes coexists with a reduction in the antioxidant status, which can increase the deleterious effects of
free radicals [20]. Since the synthesized compounds
exhibited antioxidant activity in in-vitro and in-vivo studies, they were evaluated for their antihyperglycaemic
activities in streptozotocin-induced diabetic rats. Trolox
was selected as a reference drug. Figure 3 shows that comwww.archpharm.com
52
N. Rastkari et al.
Arch. Pharm. Chem. Life Sci. 2008, 341, 49 – 54
uncorrected. 1H-NMR spectra were obtained using a FT-400 Varian Unity plus spectrometer (Varian, Switzerland). Tetramethylsilane was used as an internal standard. Mass spectra were
obtained using a Finnigan Mat TSQ-70 spectrometer at 70 eV
(Finnigan Mat, Bremen, Germany). The IR spectra were obtained
using Nicolet FTIR Magna 550 spectrographs (KBr disks) (Nicolet,
Madison, WI, USA). The purity of compounds was confirmed by
TLC using different mobile phases. Elemental analyses were carried out on a Heraeus CHN-O rapid elemental analyzer (Heraeus
GmbH, Germany) for C, H, and N and the results are within
l 0.4% of the theoretical values.
Figure 3. Effect of compounds 3a – h and trolox on rat fasting
blood sugar. (a) the difference between control and treated
groups is significant at P a 0.05; (b) compound 3a and trolox had
similar activity.
pound 3a causes a significant decrease in fasting blood
sugar (FBS). The high free radical-scavenging activity of
compound 3a may explain its significant antidiabetic
effect.
The results demonstrated that compounds with a substitution in the R2 position (3a – e and 3h) revealed good
to high RSA reducing power and lipid peroxidationinhibition values, and, in some cases, showed activities
similar to the reference drug. Antioxidative activity is a
multifactorial potential. Propensity of radical formation
and stabilization, ability of metal complexation, and lipophilicity are important factors for the antioxidant activity. The presence of an electron-donating hydroxyl or
methyl substituent in the R2 position may increase the
stability of the radical and, hence, the antioxidative activity. Compound 3a showed good activity in both in-vitro
and in-vivo experiments.
In addition, it should be noted that the order of chemical-scavenging activities using the DPPH radical is well
correlated with that of biological antioxidative activities.
More remarkable is that FRAP (Fig. 1), TBARS (Fig. 2), and
the antidiabetic activity (Fig. 3) evaluated by the concentration of blood glucose are just in the same order among
compounds 3a – h, in spite of their difference of pharmacokinetics and metabolism.
This work was supported by grants from the research council of
Tehran University of Medical Sciences and INSF (Iran National
Science Foundation).
The authors have declared no conflict of interest.
Chemistry
Substituted benzimidazolo[2,1-b]benzo[e]thiazepin-5(10H)-ones
were produced in moderate to good yield by reaction of substituted mercapto benzimidazole derivatives 1a – g [21] with 2chloromethylbenzoyl chloride 2 [22] as a coupling component
(Scheme 1). Reduction of compound 3c (R2 = NO2) with Raney
nickel in ethanol gave the corresponding amino compound 4.
Compound 4 was unstable and was used in the next step without
further purification. Diazotization of compound 4 with sodium
nitrite in the presence of concentrated sulphuric acid followed
by hydrolysis in water [23] afforded compound 3h in moderate
yield (Table 1).
Benzimidazolo[2,1-b]benzo[e]thiazepin – 5(10H)-one 3a
2-Chloromethylbenzoyl chloride (0.4 mL) was added dropwise to
a suspension of 2-mercaptobenzimidazole 1a (3 mmol) in boiling acetonitrile (50 mL). The mixture was refluxed for 3 h. The
hot solution was filtered and the filtrate was refrigerated. The
precipitate was collected and recrystallized from acetone to give
compound 3a. IR (KBr): m max cm – 1 1710 (C=O). 1H-NMR (DMSOd6): d = 4.52 (s, 2H, S-CH2), 7.38 (m, 2H, H-C7, H-C9), 7.42 (m, 2H, HC2, H-C3), 7.65 (t, 1H, H-C8, J = 7.2 Hz), 7.85 (dd, 1H, H-C1, J = 8.0 and
2.4 Hz), 7.90 (d, 1H, H-C6, J = 7.6 Hz), 7.95 (dd, 1H, H-C4, J = 8.0 and
2.4 Hz). MS: m/z (%) 266 [M+] (20), 199 (40), 157 (20), 120 (15), 104
(100). Anal. calcd. for C15H10N2OS: C, 67.65; H, 3.78; N, 10.52.
Found: C, 67.79; H, 3.60; N, 10.78.
Compounds 3b – 3g were prepared similarly (Table 1).
3-Methyl-benzimidazolo[2,1-b]benzo[e]thiazepin –
5(10H)-one 3b
IR (KBr): m max cm – 1 1720 (C=O). 1H-NMR (DMSO-d6): d = 2.42 (s,
3H, Me), 4.53 (s, 2H, S-CH2), 7.21 (d, 1H, H-C2, J = 8.0 Hz), 7.40 (m,
2H, H-C7 , H-C9), 7.66 (dt, 1H, H-C8, J = 8.0 and 2.0 Hz), 7.77 (s, 1H,
H-C4), 7.83 (d, 1H, H-C1, J = 8.0 Hz), 7.90 (dd,1H, H-C6, J = 8.0 and
2.0 Hz). MS: m/z (%) 280 [M+] (15), 265 (35), 201 (30), 157 (30), 104
(100). Anal. calcd. for C16H12N2OS: C, 68.55; H, 4.31; N, 9.99.
Found: C, 68.73; H, 4.11; N, 10.18.
3-Nitro-benzimidazolo[2,1-b]benzo[e]thiazepin – 5(10H)one 3c
Experimental
General
Chemicals were purchased from Merck Chemical Company
(Darmstadt, Germany). Melting points were determined on a
Kofler hot stage apparatus (Reichert, Vienna, Austria) and are
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2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
IR (KBr): m max cm – 1 1342 (NO2), 1470 (NO2), 1690 (C=O). 1H-NMR
(DMSO- d6): d = 4.53 (s, 2H, S-CH2), 7.41 (m, 2H, H-C7, H-C9), 7.63 (t,
1H, H-C8, J = 8.0 Hz), 7.88 (d, 1H, H-C6, J = 8.0 Hz), 7.93 (d, 1H, H-C1, J
= 8.8 Hz), 8.15 (dd, 1H, H-C2, J = 8.8 and 2.0 Hz), 8.44 (d, 1H, H-C4, J
= 2.0 Hz). MS: m/z (%) 311 [M+] (10), 254 (30), 210 (45), 169 (70), 104
(100). Anal. calcd. for C15H9N3O3S: C, 57.87; H, 2.91; N, 13.50.
Found: C, 57.95; H, 2.70; N, 13.66.
www.archpharm.com
Arch. Pharm. Chem. Life Sci. 2008, 341, 49 – 54
3-Chloro-benzimidazolo[2,1-b]benzo[e]thiazepin –
5(10H)-one 3d
IR (KBr): m max cm – 1 1700 (C=O). 1H-NMR (DMSO-d6): d = 4.50 (s,
2H, S-CH2), 7.39 (m, 2H, H-C7, H-C9), 7.45 (d, 1H, H-C2, J = 8.0 Hz),
7.60 (dt, 1H, H-C8, J = 8.8 and 2.4 Hz), 7.88 (d, 1H, H-C1, J = 8.0 Hz),
7.98 (dd, 1H, H-C6, J = 8.8 and 2.4 Hz), 8.10 (s, 1H, H-C4). MS: m/z
(%) 302 [M+ + 2] (5), 300 [M+] (15), 260 (19), 167 (45), 125 (56), 104
(100). Anal. calcd. for C15H9ClN2OS: C, 59.90; H, 3.02; N, 9.31.
Found: C, 59.82; H, 2.89; N, 9.17.
3-Bromo-benzimidazolo[2,1-b]benzo[e]thiazepin –
5(10H)-one 3e
IR (KBr): m max cm – 1 1710 (C=O). 1H-NMR (DMSO-d6): d = 4.51 (s,
2H, S-CH2), 7.35 (m, 2H, H-C7, H-C9), 7.49 (m, 2H, H-C2, H-C8), 7.60
(d, 1H, H-C1, J = 7.2 Hz), 7.88 (dd, 1H, H-C6, J = 8.8 and 2.4 Hz), 8.09
(s, 1H, H-C4). MS: m/z (%) 346 [M+ + 2] (12), 344 [M+] (14), 265 (45),
201 (34), 157 (26), 104 (100). Anal. calcd. for C15H9BrN2OS: C,
52.19; H, 2.63; N, 8.11. Found: C, 52.28; H, 2.46; N, 8.37.
2-Methyl-benzimidazolo[2,1-b]benzo[e]thiazepin-5(10H)one 3f
IR (KBr): m max cm – 1 1705 (C=O). 1H-NMR (DMSO-d6): d = 2.55 (s,
3H, Me), 4.50 (s, 2H, S-CH2), 7.18 (d, 1H, H-C3, J = 8.0 Hz), 7.39 (m,
2H, H-C7, H-C9), 7.61 (dt, 1H, H-C8, J = 8.0 and 2.0 Hz), 7.70 (s, 1H,
H-C1), 7.88 (d, 1H, H-C4, J = 8.0 Hz), 7.92 (dd,1H, H-C6, J = 8.0 and
2.0 Hz). – -MS: m/z (%) 280 [M+] (12), 264 (44), 199 (25), 147 (32),
110 (100). Anal. calcd. for C16H12N2OS: C, 68.55; H, 4.31; N, 9.99.
Found: C, 68.69; H, 4.12; N, 10.17.
2-Nitro-benzimidazolo[2,1-b]benzo[e]thiazepin – 5(10H)one 3g
IR (KBr): m max cm – 1 1340 (NO2), 1470 (NO2), 1700 (C=O). 1H-NMR
(DMSO-d6): d = 4.53 (s, 2H, S-CH2), 7.41 (m, 2H, H-C7, H-C9), 7.60 (dt,
1H, H-C8, J = 8.0 and 2.0 Hz), 7.88 (dd, 1H, H-C6, J = 8.0 and 2.0 Hz),
7.95 (d, 1H, H-C4, J = 8.8 Hz), 8.20 (d, 1H, H-C3, J = 8.8 Hz), 8.48 (s,
1H, H-C1). MS: m/z (%) 311 [M+] (15), 254 (32), 220 (25), 195 (20),
169 (54), 110 (100). Anal. calcd. for C15H9N3O3S: C, 57.87; H, 2.91;
N, 13.50. Found: C, 57.70; H, 3.16; N, 13.37.
3-Hydroxy-benzimidazolo[2,1-b]benzo[e]thiazepin –
5(10H)-one 3h
A stirring mixture of compound 3c 1 mmol) in ethanol (25 mL)
and Raney nickel (0.432 g) was refluxed for 3 h. It was filtered
hot, and the solvent was evaporated under reduced pressure to
give compound 4 which was unstable and was used without further purification in the next reaction.
Concentrated sulphuric acid (8 mL) was added cautiously to
10 mL of water contained in a 100 mL beaker, and 5 mmol
(196 mg) of 4 was added into it. An amount of 1 – 1.5 g crushed
ice was added and stirred until a homogeneous paste resulted.
The reaction mixture was cooled to 0 – 58C and stirred. A cold solution of 400 mg (5.1 mmol) of sodium nitrite in 5 mL of water
was added dropwise. Stirring was continued for 10 min to give
the diazonium salt.
While the diazotization was in progress, 10 mL of concentrated sulphuric acid was added cautiously to 15 mL of water in
a 100 mL round-bottomed flask. The mixture was heated to boiling. The diazonium solution was added dropwise from a separatory funnel supported over the flask. After the addition was com-
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2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Benzimidazolo[2,1-b]benzo[e]thiazepin-5(10H)-ones
53
pleted, the reaction mixture was boiled for further 5 min,
cooled, and poured into crushed ice. The precipitate was filtered
and recrystallized from acetonitrile to give 3h. IR (KBr): m max
cm – 1 1705 (C=O), 3433 (OH). 1H-NMR (DMSO-d6): d = 4.54 (s, 2H, SCH2), 7.40 (d, 1H, H-C2, J = 8.6 Hz), 7.52 (s, 1H, H-C4), 7.60 (m, 2H,
H-C7, H-C9), 7.72 (dt, 1H, H-C8, J = 8.6 and 2.0 Hz), 7.86 (d, 1H, H-C1, J
= 8.6 Hz), 7.93 (dd, 1H, H-C6, J = 8.6 and 2.0 Hz). MS: m/z (%) 282
[M+] (9), 280 (45), 154 (30), 145 (20), 121 (100), 69 (40). Anal. calcd.
for C15H10N2O2S: C, 63.82; H, 3.57; N, 9.92. Found: C, 63.95; H,
3.70; N, 9.71.
Antioxidant activity studies
DPPH radical scavenging activity and oxidation potential
A DPPH radical-scavenging activity of test compounds and trolox
were measured using the method of Blois with a slight modification. A 0.1 mM solution of 1,1-diphenyl-2-picrylhydrazyl (DPPH)
in methanol was prepared and 1 mL was added to 3 mL of sample solutions in methanol at a range of concentrations (0.0156 –
0.25 g/L). After 30 min, the absorbance was measured at 517 nm.
All data are an average of triplicate analyses. Decreasing of the
DPPH solution absorbance indicates an increase of the DPPH radical-scavenging activity. This activity is shown as%DPPH radicalscavenging that is calculated by the equation:
% DPPH radical scavening activity ¼
control absorbance sample
control absorbane
6100
The DPPH solution without a sample solution was used as control.
Reducing power assay in serum
In the in-vivo study protocol, male Sprague – Dawley rats weighing 120 – 180 g were randomly distributed into 10 groups with
six animals in each group. The groups were divided into control,
trolox-treated, and test-compound-treated (eight different
groups). The animals in the treated groups were orally administered 100 mg/day/kg body weight of test compounds and trolox
(test compounds and trolox were dissolved in 6 mL DMSO and
the volume adjusted to 1000 mL with water). The control animals received only the mixture of solvents to observe solvents
effect as gavage vehicle. Trolox was selected as a reference drug
in the in-vivo experiments. All treatments were continued for
ten days after which blood samples were collected following an
overnight fast.
The antioxidant capacity of plasma was determined by measuring its ability to reduce Fe3+ to Fe2+ established as FRAP test and
was described previously by Benzie and Strain. The complex
between Fe2+ and 2,4,6-tripyridyl-1,3,5-triazine (TPTZ) gives a
blue color with absorbance at 593 nm.
Lipid peroxidation assay in serum
Pre-treatment of animals was explained in previous section. Thiobarbituric acid reactive substances (TBARS) measurement was
used to measure the rate of lipid peroxidation. In this method,
plasma samples were mixed with trichloroacetic acid 20% (w/v)
and the precipitate was dispersed in H2SO4 (0.05 M). 2-Thiobarbituric acid (0.2% in 2 M sodium sulfate) was added and heated for
30 min in a boiling water bath. TBARS adducts were extracted by
n-butanol (4 mL) and absorbance was measured at 532 nm. The
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N. Rastkari et al.
calibration curve of a 1,1,3,3-tetrahydroxypropan standard solution was used to determine the concentrations of TBARS in samples.
Antidiabetic activity studies
For purposes of assessing the antidiabetic effect, male Spargue –
Dawley rats weighing 180 – 200 g were randomly distributed
into control and treated groups (trolox and test compounds),
with six animals in each group. Diabetes was induced in both
groups by a single i.p. injection of 75 mg/kg of streptozotocin
freshly dissolved in saline. The treated animals received 100 mg/
day/kg body weight of test compounds and trolox (test compounds and trolox were dissolved in 6 mL DMSO and the volume
adjusted to 1000 mL with water). The control animals received
only the mixture of solvents to observe solvents effect as gavage
vehicle. Trolox was selected as a reference drug. All treatments
were continued for 10 days. Blood sugar was measured in blood
samples obtained from the tail of the rats using a diagnostic kit.
The rat's average blood glucose was 70 – 80 mg/dL prior to the
experiment and increased to 300 – 350 mg/dL after single i.p.
injection of 75 mg/kg of streptozotocin. Water intake and urination were also drastically increased.
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