Sci.Int.(Lahore),28(2),1233-1236,2016
ISSN 1013-5316; CODEN: SINTE 8
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IN-VITRO ANTIOXIDANT BIOASSAY OF SYNTHESIZED BIPHENYL
ANALOGUES
*
Qamar Ali1, Asad Gulzar1, Abrar Hussain1, Amina Asghar1, Ikram Ullah Bajwa1
Division of Science and Technology, University of Education, Township Campus, Lahore, Pakistan.
*Corresponding author: Dr. Qamar Ali
e-mail:[email protected]
ABSTRACT- Two different series of synthetic biphenyl analogues were synthesized and evaluated for in-vitro antioxidant
bioassay. Two different antioxidant bioassays were carried out (a) in vitro DPPH free radical scavenging bioassay and (b) in
vitro superoxide anion scavenging bioassay. Only compound 2a was found to show moderate activity against DPPH bioassay,
while compounds 4a and 4b were found to be good scavengers of superoxide anion radical. Compounds 2a and 2b were found
to be moderately active on this assay while compounds 2c-e were found to be inactive as antioxidants.
Keywords: Biphenyl analogues, antioxidant bioassay, DPPH, anion radical scavengers, % RSA values .
INTRODUCTION
Living systems undergo generation of free radicals from
normal metabolic processes. Free radicals play a crucial role
in living organisms [1]. For example, oxygen radicals exert
very important actions in living cells, including signal
transduction, gene transcription and regulation [2-3].
However, free radicals speed up oxidation of biomolecules,
stimulate glycation of protein, inactivate enzymes and cause
alteration in the structure and function of membranes leading
to cell injury and death [1]. These metabolic processes may
result a variety of fatal diseases like diabetes, cancer,
atherosclerosis, stroke, and Alzheimer’s disease [4-10, 11].
Antioxidants are the substances having ability to scavenge
free radicals and to protect the body from free radical induced
oxidative stress of biomolecules. Antioxidants deactivate free
radical by suppressing formation of free radicals by reducing
hydrogen peroxide, binding to the metal ions and quenching
singlet oxygen and superoxide [12]. Because of their
superficial effects for health promoting, including
anticarcinogenic, antiatherogenic, anti-ulcer, antithrombotic,
antiinflammatory, immunomodulating, antimicrobial and
analgesic effects, antioxidants have become a focus of
research interest [13]. So the search for utilization of
antioxidants has greatly improved in recent years. Synthetic
antioxidants commonly used as food additives are butylated
hydroxytoluene, butylated hydroxyanisole, and tertbutylhydroquinone. These synthetic antioxidants are
functional at very low concentrations of a 0.01 g/100 g
sample or less and are relatively cheap so they are oftenly
used [14]. Keeping all these facts in view, synthetic biphenyl
analogues were evaluated for antioxidant activity.
MATERIALS AND METHODS
In order to investigate the difference in activity of
synthesized biphenyl analogues, two different classes of
compounds were synthesized from [1,1ʹ-biphenyl]-2,2ʹ-diol
and Fast blue B salt.
General Procedure for the Synthesis of Biphenyl
Analogues Based on [1, 1ʹ-biphenyl] - 2, 2ʹ-diol
The synthesis of biphenyl analogues derived from 1 was
achieved through the route outlined in Scheme 1.
After dissolution of compound 1 in 15 mL of acetone, 1.5 g
(10.21 mmol) of K2CO3 was added. After stirring for 30
minutes, 2 mL (13.4 mmol) of tert-butyl bromoacetate was
99).
added. After 3 hours, 1N HCl was added to quench the
reaction. The required product was 2a extracted with 30 mL
of CH2Cl2 and purified through column chromatography
giving 80% yield. Compound 2b was obtained by treating 2a
with TFA for 1 hour, followed by work up with hexane and
toluene giving 90% yield. Compound 2c was obtained from 1
by refluxing with 1 mL (13.4 mmol) methyl chloroformate in
the presence of acetone for 36 hours affording 80 % yield.
Compound 2d was obtained by refluxing 1.3 mL of
dibromomethane (13.4 mmol) with 1 affording 45 % yield,
while compound 2e was obtained by treating statistical
amount of dibromomethane with 1 and K2CO3 in ethanol
acting as solvent affording 50 % yield.
General Procedure for the Synthesis of Biphenyl
Analogues based on Fast Blue B salt 3
The synthesis of biphenyl analogues 4a-b was carried out
from compound 3 through the route outlined in Scheme 2.
After dissolution of 28 g (0.17 mmol) of KI in 200 ml of
water, 10.00 g (21 mmol) of 3 was added and the reaction
was carried out for 12 hours at room temperature. The crude
reaction product was extracted by adding 30 mL
dichloromethane three times. The crude mixture was purified
through column chromatography. The solvent system used
for silica gel column was dichloromethane: hexane (1:4)
resulted fine crystals of 4a giving 70 % yield. In addition to
4a, fine crystals of 4b were also obtained giving 25% yield.
ANTIOXIDANT EVALUATION OF SYNTHESIZED
BIPHENYL ANALOGUES
1. In vitro DPPH (1,1-diphenyl-2-picryl hydrazyl) free
radical scavenging bioassay
DPPH was used for calculation of free radical scavenging
activity of synthesized biphenyl analogues [15- 16]. The
ethanolic solution of 5 µL (1 mM in DMSO) of the
compound to be tested and 95 µL of DPPH (3 mM) was
taken into a 96-well microliter. The reaction mixture was
incubated at 37 oC for 30 minutes and was monitored by
taking absorbance at 515 nm on microtiter reader.
2. In vitro superoxide anion scavenging bioassay
The synthesized biphenyl derivatives were screened by the
technique used by Gaulejac et al., in 1999 [17]. The reagents
used for making the reaction mixture were consisted of 40 µL
of 280 µM β- (NADH), 40 µL 280 µM of NADH, 80 µM of
(NBT), 20 µL of 80 µM phenazine methosulphate (PMS) 10
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Sci.Int.(Lahore),28(2),1233-1236,2016
R
OH
O
OH
O
1
R'
2a-e, R = -COOt-Butyl, -COOH, -COOMe CH2Br, -CH2Br
R' = -COOt-Butyl, -COOH, -COOMe, CH2Br, H
Scheme 1: Synthesis of [1, 1'-biphenyl] - 2, 2'-diol analogues, 2a-e.
H3C
H3C
O
O
Cl ZnN
N
N
R'
N ZnCl
O
O
CH3
R
CH3
3
4a-b, R = I, I
R' = I, H
Scheme 2: Synthesis of Fast blue B salt analogues 4a-b.
Table 1: Results of superoxide assay of biphenyl analogues.
Compound
2a
2c
2e
4b
%RSA (Radical
Scavenging
Activity)
65
9
16
87.3
Compound
2b
2d
4a
%RSA (Radical
Scavenging
Activity)
53
43
71.5
Figure - 1: Results of superoxide assay of biphenyl analogues
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Sci.Int.(Lahore),28(2),1233-1236,2016
ISSN 1013-5316; CODEN: SINTE 8
µL of 1 µM sample and 90 µL of 0.1 M phosphate buffer
(pH 7.4). The sample was prepared in DSMO. A 96-well
microtitre plate was used to carry out the reaction at room
temperature. The absorbance was taken at 560 nm. The
superoxide formation was checked by analysis water soluble
blue Formazan dye. A lower absorbance of reaction mixture
revealed a higher scavenging activity of sample. % RSA of
test compounds was determined in comparison to a control by
using following equation:
% RSA = 100- {(OD test compound/ OD control) x 100}
RESULTS AND DISCUSSION
In DPPH free radical scavenging bioassay, % RSA was
determined by taking comparison with control containing
BHA (3-t-Butyl-4-hydroxyanisole) dissolved in DMSO. Only
compound 2a was found to show moderate activity against
DPPH bioassay showing 44.3 % RSA, while none of
remaining synthesized biphenyl analogues showed any
reasonable activity against DPPH bioassay.
In superoxide assay of biphenyl analogues, Quercetin was
used as a standard. 4, 4′-Diiodo-3, 3′-dimethoxybiphenyl (4a)
and 4-iodo-3, 3′-dimethoxybiphenyl biphenyl (4b) were
found to be good scavengers of superoxide anion radical
showing 71.5 and 87.3 % RSA values respectively. Di-tertbutyl 2, 2′-(biphenyl-2, 2′-diyldioxy) diacetate (2a) and 2,2ʹ[biphenyl-2,2ʹ-diylbis(oxy)]diacetic acid (2b) were found to
be moderately active showing 65 and 53 % RSA values
respectively. While compounds 2c-e were found to be
inactive in superoxide bioassay.
Synthesis of a series of biphenyl analogues 2(a-e) and 4(a-b)
is illustrated in Scheme 1 and Scheme 2. Biphenyl analogues
were synthesized by using general synthetic route involving
nucleophilic substitution reactions on functional biphenyl
analogues.
Synthetic biphenyl analogues were evaluated for antioxidant
bioassay. Compound 2a was the only biphenyl analogue
showing moderate activity against DPPH bioassay showing
44.3 % RSA (Radical Scavenging Activity), while
compounds (4a-4b)) were found to be good scavengers of
superoxide anion radical showing due to higher % RSA
values. Compounds (2a-b) were found to be moderately
active showing average % RSA values, respectively.
However, compounds (2c-e) were found to be inactive in
superoxide bioassay showing very low % RSA values.
CONCLUSION
Synthesis of a series of biphenyl analogues 2(a-e) and 4(a-b)
is illustrated in Scheme 1 and Scheme 2. Biphenyl analogues
were synthesized by using general synthetic route involving
nucleophilic substitution reactions on functional biphenyl
analogues.
Synthetic biphenyl analogues were evaluated for antioxidant
bioassay. Compound 2a was the only biphenyl analogue
showing moderate activity against DPPH bioassay showing
44.3 % RSA (Radical Scavenging Activity), while
compounds (4a-4b)) were found to be good scavengers of
superoxide anion radical showing due to higher % RSA
1235
values. Compounds (2a-b) were found to be moderately
active showing average % RSA values, respectively.
However, compounds 2c-e were found to be inactive in
superoxide bioassay showing very low % RSA values.
ACKNOWLEDGEMENTS
The authors are indebted to the financial support provided by
the HEC (Higher Education Commission, Pakistan) and HEJRIC, University of Karachi for providing necessary space and
facilities for bioassays.
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