CHAPTER 7 Evaluation of Biological...
30
CHAPTER 7 Evaluation of Biological Activities
Transcript of CHAPTER 7 Evaluation of Biological...
CHAPTER 7
Evaluation of Biological Activities
CHapter 7 'EvaCuation of (BiolbgicaC activities
7.1. Introduction
'The most fruitful basis for the discovery of a new drug is to start with an old
drug.'- Sir James Whyte Black, the winner of 1988 Nobel Prize in physiology and
medicine [1].
Nitrogen heterocycles are of immense importance not only biologically and
industrially but also to the functioning of any developed human society as well. The
majority of pharmaceutical products that mimic natural products with biological
activity are heterocycles. Therefore, researchers are on a continuous pursuit to design
and produce better pharmaceuticals, pesticides, insecticides, rodenticides, and
weedicides by following natural models. Heterocyclic compoimds, especially nitrogen
heterocycles, are most important class of compounds in the pharmaceutical and
agrochemical industries, in which heterocycles comprising around 50-60% are
covered as a drug substances. N-heterocycles such as imidazoles, triazoles, thiazoles,
oxadiazoles, thiadiazoles pyrroles, indoles, and carbazoles are important structural
motifs and are present in an extensive number of biologically active compounds.
6-Membered aromatic rings containing two nitrogen atoms, such as phthalazinones,
quinazolinones, pyrimidines and pyrimidinones, possess a broad spectrum of
biological activities and are therefore of interest as target compounds in
pharmaceutical and medicinal chemistry.
During recent years, there have been intense investigations on imidazo[2,l-
6][l,3]thiazole and imidazo [2,1-6][1,3] benzothiazole moiety many of which are the
important heterocyclic nucleus which has been used extensively in medicinal
chemistry. The imidazo[2,l-fe][l,3]thiazole derivatives possess versatile biological
properties such as diuretic [2], antitumor [3], anti-inflammatory [4],
immxmosuppressive [5,6], antimicrobial antitubercular [7,8], and analgesic activity
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CHapter 7 'EvaCuation of (BiologkaC activities
[9,10]. Further imidazo[2,l-6][l,3]thiazole and imidazo[2,l-6][l,3]benzothiazole also
displays pharmacological activity [11-13]. Compounds containing imidazo[2,l-
6][l,3]tliiazole and imidazo[2,l-6][l,3] benzothiazole moiety have been reported to
exhibit anti-inflammatoiy, antimicrobial and cardiotonic selective agonist properties
for the human orphan nuclear receptor CAR (constitutive androstane receptor) and
diuretic agents [14-17].
The development of potent and effective antimicrobial agent is most important to
overcome the emerging multi-drug resistance strains of bacteria and fimgi such as
methicillin resistant staphylococcus aureus (MRSA) [18, 19].
The heterocyclic compounds play significant role in developing new
antimicrobial, anticancer, antimalarial, anticonvulsant agents. Recent observations
suggested that, heterocyclic compounds containing nitrogen are very important
class of organic heterocycles, because of their wide appUcation in medicine,
agriculture and technology aspects. Among these, 2-phenylimidazo[l,2-a]pyridine
derivatives are of significant synthetic interest due to their diverse range of
biological activities, some of them showed pharmacological properties such as
anti-inflammatory [20,21], aromatase-inhibitors [22], antibacterial [23], antifungal
[24], antiviral [25] and analgesic [26] activities.
Azole class of drugs particularly fused imidazoles occupy prominent place in
medicinal chemistry because of their broad spectrum pharmacological activities such
as anti-inflammatory, analgesic, anticancer, antimicrobial, antiviral, pesticidal
cytotoxicity and anti-arrhythmic [27-30] activities. Omeprazole, Mebendazole,
Pimobendan, and Albendazole are well known drugs in the market which contain
fused imidazole as active core moiety.
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CHapter 7 <EvaCuation of (BiohgicaC activities
In light of these facts, the different heterocyclic compounds synthesized in the
present investigation have been evaluated for the following biological activities.
1. Antimicrobial activity
a. Antibacterial
b. Antifungal
7.2. Antimicrobial activity
7.2.1. Antibacterial and antifungal activity
Antibacterial resistance is a global clinical and public health problem that has
emerged with alarming rapidity in recent years and imdoubtedly will increase in the
near future. The morbidity, mortality, and financial costs of such infections pose an
increasing burden for health care systems worldwide. Life threatening infectious
disease caused by multidrug-resistant pathogenic bacteria augmented an alarming
level aroimd the world. Owing to this increased microbial resistance, new classes of
antimicrobial agents with novel mechanisms are todays need to fight against the
multidrug-resistant infections [31]. Heterocyclic compounds play an important role in
an imtiring effort aimed at developing new antimicrobial agents.
Since resistance of pathogenic bacteria towards available antibiotics is rapidly
becoming a major worldwide problem, the synthesis and design of novel compoimds
to deal with resistant bacteria has become one of the most important areas of
antibacterial research today.
The bacterial diseases are dangerous to human beings [32, 33]. Staphylococcal
infections are among the most conmion bacterial infections and range from the trivial
to the fatal disease and some of the staphylococcal infections are folliculitis, fruncle
(boil), wound infection on skin and soft tissue, osteomylitis, arthritis on
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CHapter 7 <EvaCuation of (BiobgicaC activities
musculoskeletal, toncilitis, pharyngitis, sinusitis and bronchopneumonia related to
respiratory tract infections.
Bacillus are common in both aerobic and anaerobic inhabitat and can cause
deadly diseases like anthrax, now it has been brought imder control by using
antibiotics and these cause food poisoning leading to stomach ache, headache,
vomiting, diarrhea [34].
The bacteria like Escherichia coli and Klebseilla pneumonia are the gram
negative bactreria and belong to Enterobactriaceae class. They cause many clinical
infections like urinary tract infections and diarrhea by producing enterotoxin (toxin to
gastrointestinal tract). The toxin causes dehydration and salt imbalance, which is
substantial enough to be life threatening in infants. The Klebsiella pneumonia is the
sub-species of Aerobacter aerogenes. It is the second popular member of the aerobic
bacterial flora of the human intestine. It causes pneimionia, urinary tract infections,
pyogenic infections and septicmia rarely. Klebsiella pneumonia causes serious
diseases with high case of fatality. It occurs in middle age or older persons who have
medical problems such as alcoholism, chronic bronchopuhnonary disease or diabetes
mellitus [35].
Our body is an ecosystem to millions of bacteria, fimgi and other microbes
that naturally coexist in the skin, digestive tract and other regions. Immuno
compromised patients like HIV patients are prone for more opportunistic infections
by normal microflora as well as invasive fungi. Many research groups are working
aroimd the world to understand and prevent these infections in normal and
immunologically suppressed individuals. Candida albicans is one of the normal flora
that can become a potent pathogen taking advantage of immunologically suppressed
status of individuals and causes the most prevalent disease called candidiasis.
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Cfiapter 7 %va[ua.tion of (BiobgicaC activities
Different classes of organic compounds such as azoles, allylamine derivatives,
lipopeptides, polyene derivatives, N-hydroxy-2-pyridones and fluorinated pyrimidines
are known for treatment against fimgal infections [36]. These antifungal drugs have
some limitations such as higher toxicity to the host tissues, drugs relapse development
of drug resistant strains and high cost [37, 38]. Thus, there is an immediate need for
development of newer antifungal agents.
It is evident from the above facts that several compoimds possessing thiazole,
benzimidazole and oxadiazole nucleus are capable of exhibiting antibacterial activity.
When these ring systems are fused or coupled with other heterocycle directly or
through bridges, the resulting compoimds may exhibit enhanced antibacterial
property. All the heterocyclic compounds synthesized in the course of present
investigation were screened for antibacterial activity.
Evaluation of antibacterial and antifungal activity
The antibacterial and antifungal activity of synthesized compoimds was
studied comparatively with that of standard drugs Ciprofloxacin, Ampicillin
(antibacterial) and fluconazole (antifungal). The preliminary investigation was done
by using agar well diffusion method [39]. Further some of the compounds which
showed good zone of inhibition in primary screening were assessed by minimum
inhibitory concentration (MIC) using serial broth-dilution method (National
committee for clinical laboratory standards (NCCLS) 1982) [40] at different
concentrations i. e; 1, 10, 25, 50,100 and 150 ng/mL to quantify the antimicrobial
potency of the compounds against bacterial strains Escherichia coli. Staphylococcus
aureus, Pseudomonas aeruginosa, Bacillus subtilius, Salmonella typhi.Psedomonas
syringae, Klebsiella pneumoniae and fungal strains Aspergillus flavous,
Chrysosporium keratinophilum, Candida albicans, Microspora griseous, Aspergillus
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Cfiapter 7 'EvaCuation of <BiolJogica[ activities
terns. The above different microbial strains used in the present work were procured
and all anti-microbial activity work has been carried out in the Department of
Biochemistry and Botany, Kuvempu University.
Antibacterial activity
Materials and Method:
The antibacterial activity of test compoimds was screened against the above
mentioned strains and the following materials were used for the testing
1. Nutrient agar
2. Sterilized petri dishes
3. Potato dextrose broth
4. Tuberculin syringes with needles
5. Sterile test tubes for the preparation of solutions of test compounds in desired
concentrations.
Preparation of media
Nutrient agar was prepared by dissolving bacteriological peptone (1.0%), meat
extract (0.5 %), and sodium chloride (0.5 %) in distilled water. The pH of the solution
was adjusted to 7.0 -7.4 by using sodixmi hydroxide (40 % approximately, 0.25 ml for
100 ml of nutrient broth) and then sterilized for 30 minutes at 15 lbs pressure in an
autoclave.
Preparation of sub-culture
One day prior to the test, microorganisms were inoculated into sterilized
o
nutrient broth tubes and incubated at 37 C for 24 h. After incubating the broth
obtained was used as inoculimis for the test.
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CAapter 7 'Evaluation of (BiolbgicaC activities
Antifungal activity
Materials and Method
The following materials were used.
1. Potato dextrose agar
2. Micropipette
3. Sterilized petri dishes
4. Potato dextrose broth (48 h old)
5. Tuberculin syringes with needles
6. Sterile test tubes for the preparation of solutions of test compounds in desired
concentrations.
Preparation of media
Potato dextrose agar was prepared by dissolving potato dextrose agar (20 g) in
distilled water (500 mL); the pH of the solution was adjusted to 5.6 and then sterilized
for 15 minutes at 121 °C at 15 lb pressure in an autoclave.
Preparation of sub-culture
Two days prior to the test, the microorganisms were inoculated into sterilized
potato dextrose broth and incubated at 25 °C for 72 h. After incubating the same for
two days, the broth obtained was used as iimoculum for the test.
Preparation of solutions
The solutions of all synthesized compounds were prepared in
Dimethylsulfoxide (DMSO) and tested at the concentration of 100 |il/mL of solution.
Methods of testing
Agar well diffusion method
The antibacterial and antifungal activity was carried out by agar-well diffusion
method which is a simple susceptibility screening method. The method depends on
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CHapter 7 'EvaCuation of (BiotbgicaC activities
the diffusion of antibiotic from a cavity through the solidified agar layer in a petri dish
to an extent such that growth of the added microorganism is prevented entirely in a
circular area or zone around the cavity containing a solution of antibiotic.
The previously liquefied medium was inoculated appropriate to the assay with
the requisite quantity of the suspension of the microorganisms between 40-50°C and
the inoculated medixmi was poured into petri dishes to give a depth of 3-4 mm. Ensure
that the layers of medium were uniform in thickness by placing the dishes on a
leveled surface.
The dishes thus prepared were stored in a manner so as to ensure that no
significant growth or death of the test organism occurs before the dishes were used
and the siuface of the agar layer was dry at the time of use. Each microorganism was
suspended in nutrient broth and diluted approximately to colony forming imit
(cfij/mL). They were 'flood-inoculated' onto the surface of nutrient agar and then
dried. Five millimeter diameter wells were cut from the agar using a sterile cork-borer
and 100^L of the test compound solution were delivered into the wells.
The dishes were left standing for 2 h at room temperature as a period of pre
incubation diffusion to minimize the effects of variation in time among the
applications of different solutions. Plates inoculated with the bacteria were incubated
at 37 °C for 24 h and the fungal culture was incubated at 25 °C for 72 h. The zone of
inhibition developed, if any, was then measured accurately. Each zone of inhibition
recorded was the average of three measurements.
Determination of Minimum inliibitory concentration (MICs)
Serial broth-dilution method
The antibacterial potency of selected series of compoimds was quantified by
determining the minimum inhibitory concentrations. The minimal inhibitory
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Cfiapter 7 'Evafuation of (Biobgicaf activities
concentrations (MICs) were determined by macro broth dilution technique according
to the National Committee for Clinical Laboratory Standards (NCCS) in three
separate experiments (1999). One of the earliest antimicrobial susceptibility testing
methods was the macro broth or tube-dilution method. This procedure involved
preparing two-fold dilutions of antibiotics (e.g., 1,10,25, 50,100 and 150 ^g/mL) in a
liquid growth medium dispensed in test tubes. The antibiotic-containing tubes were
inoculated with a standardized bacterial suspension of 1-5x10^ cfia/mL. Following
overnight incubation at 35 °C, the tubes were examined for visible bacterial growth as
evidenced by turbidity. The lowest concentration of antibiotic that prevented growth
represented the minimal inhibitory concentration (MIC).The code used for different
strains is given below.
E.c-Escherichia coli, S.a-Staphylococcus aureus, f.a-Pseudomonas aeruginosa, B.s
-Bacillus subtilius, S.t-Salmonella typhi, P.s-Psedomonas syringae, K.p-Klebsiella
pneumoniae and fungal strains AA-Aspergillus flavous, Ck-Chrysosporium
keratinophilum, Csk-Candida albicans, M.g-Microspora griseous, A.t-Aspergillus
terns.
The antibacterial and antifungal activity data of standard, control and test
compounds is given in the following tables.
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Cfiapter? T.vduation of (BiobgicaC activities
Table 7.2.2
Antimicrobial activity data of imidazopyridine derivatives (26a-o)
CI
26(a-o)
C o m p
No
.
26a
26b
26c
26d
26e
26f
26g
26h
26i
26j
26k
261
26 m
26n
26o
Diclofenac
Fluconazol
5.a±
S.D
28±0.20
30±0.10
lOiO.lO
29±0.20
12±0.30
--
19±0.30
29±0.30
25±0.30
20±0.30
08±0.30
10±0.30
14±0.30
13±0.30
08±0.30
34±0.30
Zone of inhibition in
Antibacterial
K.p
±S.D*
lOiO.lO
28±0.10
24±0.30
--
20±0.10
--
18±0.30
17±0.30
17±0.30
17±0.10
06±0.20
07±0.30
12±0.30
lliO.lO
09±0.20
32±0.30
5.14:
S.D-
08±0.30
23±0.20
22±0.30
05±0.30
20±0.10
--
17±0.30
14±0.20
22±0.20
16±0.20
04±0.10
--
14±0.10
20±0.20
19±0.10
28±0.20
E.Ck
S.D*
--
10±0.40
15±0.10
03±0.50
19±0.40
-
18±0.10
16±0.10
14±0.20
14±0.20
04±0.20
06±0.10
12±0.20
11±0.20
12±0.20
30±0.20
m m (mean:
P.a±
S.D*
07±0.20
15±0.20
21±0.50
lOiO.lO
18±0.20
03±0.20
16±0.10
15±0.50
15±0.20
10±0.20
02±0.30
10±0.50
16±0.20
11±0.20
15±0.30
24±0.20
tS.D.) n = 3
5.p±
S.D*
17d=0.30
--
16±0.50
lliO.lO
18±0.20
13±0.20
13±0.10
19±0.20
12±0.20
12±0.50
15±0.20
03±0.10
12±0.20
18±0.10
17±0.20
20±0.10
Antifungal
M ^
S.D*
25±0.50
09±0.10
17±0.10
03±0.20
--
05±0.30
--
--
--
--
--
-
--
--
--
~
34±0.30
C.a±
S.D*
06±0.20
08±0.50
19±0.20
08±0.20
20±0.10
04±0.30
12±0.20
lOiO.lO
12±0.10
09±0.20
03±0.10
03±0.10
lOiO.lO
09±0.20
03±0.10
~
22±0.30
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CHapter 7 'EvaCuation of (BiobgicaC activities
Table 7.2.3
Antimicrobial activity data of 6-(phenyl)[l,2,4]triazolo[l,5,a]pyridine derivatives
31(a-m)
Compound
31a
31b
31c
31d
31e
31f
31g
31h
31i
31j
31k
311
31m
AmpicilUn
Flucanozole
S. aureus
100
28
24
12
16
22
24
23
12
10
12
08
24
20
30
50
ML
16
13
08
12
18
20
18
—
~
"
~
20
18
24
K.pneumo
na
100
HL
18
12
10
22
18
16
28
08
08
~
08
22
30
32
50
^L
13
"
"
16
12
06
24
"
"
"
"
19
24
28
31(a-m)
P.aerugino s
100
ML
25
22
12
18
12
18
36
10
—
14
—
26
20
28
50
HL
18
08
~
15
—
12
26
~
~
06
~
18
16
20
E.feltis
100
ML
28
20
14
24
24
24
24
~
12
—
08
28
26
28
50
HL
16
14
06
18
19
16
14
~
~
~
~
14
18
24
E.Coli
100
HL
26
22
-
21
22
20
23
~
14
~
14
12
15
31
50n
L
15
12
~
12
15
16
17
~
10
—
06
—
12
28
A.Nger
100
HL
16
—
12
~
20
12
22
~
~
25
16
14
14
—
34
50n
L
08
~
-
~
14
06
16
~
—
16
12
08
08
~
28
T. Viradae
100
HL
12
08
14
~
25
20
21
~
~
24
20
16
13
—
30
50
ML
08
~
08
~
18
14
14
~
—
16
18
10
07
~
28
Each value is expressed as mean±SD of three replicates for zone of inhibition
193
CHapterZ 'Evaluation of (BioCbgicaC activities
Table 7.2.4
In vitro antibacterial activity data of synthesized compounds 30(a-e) and 32(a-h)
30(a-e)
Comp
30a
30b
30c
30d
30e
32a
32b
32c
32d
32e
32f
32g
321i
Ciprofloxacin
S.A.
17±0.2
11±0.3
16±0.2
18±0.1
17±0.2
20±0.3
19±0.2
~
21±0.2
~
17±0.1
~
19±0.3
23±0.2
32(a-h) Zone of inhibition (mm)
K.P.
13±0.3
~
15±0.2
16±0.2
17±0.1
18±0.2
17±0.1
12±0.2
17±0.2
lliO.l
13±0.2
10±0.3
20±0.1
21±0.2 ..
P. A.
14±0.3
~
12±0.2
14±0.2
18±0.1
14±0.2
15±0.3
~
~
~
15±0.2
~
12±0.1
19±0.2
S. T.
14±0.2
10±0.2
13±0.3
-
16±0.2
ISiO.l
19±0.2
11±0.2
ISiO.l
10±0.2
12±0.3
~
~
24±0.2
S.P.
16±0.2
~
11±0.3
16±0.2
14±0.1
16±0.2
18±0.1
~
14±0.2
10±0.2
17±0.3
12±0.2
16±0.1
22±0.2
E.C.
12±0.2
12±0.1
11±0.2
15±0.2
14±0.3
17±0.2
14±0.3
10±0.2
13±0.2
~
lOiO.l
~
11±0.2
26±0.2
194
Cfutpter? 'Evaluation of <BiolJogica[ activities
Table 7.2.5
In vitro antifungal activity data of synthesized compounds 30(a-e) and 32(a-Ii)
Comp
30a
30b
30c
30d
30e
32a
32b
32c
32d
32e
32f
32g
32h
Fluconazole
Zone of inhibition (nun)
A.F.
—
09±0.2
05±0.1
~
06db0.2
~
07±0.2
~
~
07±0.2
09±0.1
07±0.2
06±0.2
10±0.2
C.A.
10±0.2
19±0.2
~
~
~
~
lOiO.l
~
~
17±0.2
18±0.1
10±0.3
16±0.1
20±0.2
M G.
17±0.2
19±0.2
lOiO.l
15±0.2
12±0.3
~
14±0.1
~
—
17±0.2
18±0.1
16±0.1
18±0.1
20±0.4
A.T.
06±0.2
13±0.2
lOiO.l
15±0.2
17±0.2
~
12±0.1
~
~
15±0.2
17±0.1
15±0.3
ISiO.l
20±0.4
C.K.
12±0.1
10±0.3
~
~
~
~
09±0.2
~
~
13±0.1
12±0.2
~
~
14±0.2
195
chapter 7 'EvaCuation of (BioCogicaC activities
Table 7.2.6
Minimal inhibitory concentration (MIC, M (Molar)) data of synthesized
compounds 30(a-e) and 32(a-h)
Comp
30a
30b
30c
30d
30e
32a
32b
32c
32d
32e
Std"
Std"
Antibacterial
S.A
25.9
30.8
27.7
23.8
29.9
22.9
22.2
21.5
23.8
16.8
18.1
~
K.P
—
—
—
—
~
20.1
~
24.6
~
~
9.0
~
P. A
29.6
—
31.7
17.8
25.6
~
~
~
27.2
~
9.0
—
S. T
—
39.6
~
26.7
~
~
28.6
~
~
14.4
12.0
~
S.P
~
~
~
~
29.9
~
~
—
~
~
27.1
~
E.C
37.0
35.2
39.6
26.7
25.6
10
25.4
30.7
34.0
~
12.0
~
Antifungal
A.F
18.5
26.4
31.7
23.8
25.6
20.1
28.6
24.6
23.8
~
~
13.0
C.A
~
~
27.7
~
~
22.9
~
15.3
~
~
~
13.0
M.G
--
39.6
27.7
26.7
38.4
~
25.4
—
27.2
—
~
19.8
A.T
—
26.4
~
26.7
34.1
~
~
27.6
~
19.2
~
16.3
C.K
—
30.8
~
—
34.1
—
~
~
~
21.6
~
13.0
Std* - Ciprofloxacin ; Std**- Fluconazole
196
CHapter? 'EvaCuation of (BiolbgicaC activities
Table 7.2.7
Comp
No
29a
29b
29c
29d
29e
29f
29g
29h
29i
29j
29k
291
29m
29n
29o
Diclofenac
Fluconazole
Zone of inhibition in m m (mean ±S.D.) n = 3
Antibacterial
S.a± S.D
12±0.20
14±0.10
28±0.10
04±0.20
24±0.30
-
19±0.30
19±0.30
18±0.30
16±0.30
08±0.30
10±0.30
15±0.30
15±0.30
05±0.30
34±0.30
K.p
±S.D*
lOiO.lO
lliO.lO
24±0.30
-
20±0.10
--
18±0.30
17±0.30
17±0.30
13±0.10
06±0.20
07±0.30
12±0.30
lliO.lO
04±0.20
32±0.30
5. <±
S.D*
08±0.30
10±0.20
22±0.30
03±0.30
20±0.10
--
17±0.30
~
—
15±0.20
04±0.10
~
14±0.10
12±0.20
04±0.10
28±0.20
E.c±
S.D*
--
10±0.40
23±0.10
03±0.50
19±0.40
--
18±0.10
16±0.10
14±0.20
13±0.20
04±0.20
06±0.10
12±0.20
11±0.20
03±0.20
30±0.20
Antifungal
P.a±
S.D
07±0.20
07±0.20
21±0.50
iOiO.lO
18±0.20
03±0.20
16±0.10
15±0.50
15±0.20
10±0.20
02±0.30
07±0.50
09±0.20
09±0.20
02±0.30
24±0.20
S.p±
S.D*
10±0.30
--
16±0.50
05±0.10
15±0.20
01±0.20
13±0.10
11±0.20
~
~
~
03±0.20
~
~
—
20±0.10
M.g±
S.D*
07±0.50
09±0.10
27±0.10
03±0.20
~
05±0.30
--
-
—
07±0.50
lOiO.lO
09±0.50
~
—
—
34±0.30
Coil S.D*
06±0.20
08±0.50
15±0.20
08±0.20
14±0.10
04±0.30
12±0.20
lOiO.lO
12±0.10
09±0.20
03±0.10
03±0.10
lOiO.lO
09±0.20
03±0.10
22±0.30
Antimicrobial activities of synthesized compounds imidazopyrdine morpholine
derivatives 29(a-o)
29(a-o)
197
CHapter 7 'EvaCuation of (BiotogicaC activities
Table 7.2.8
Antibacterial activity data of compounds 19(a-c) and 22 (a-g)
Comp
19a
19b
19c
22a
22b
22c
22d
22e
22f
22g
streptomycin
E-coli
1
5.4±0.3
0
5.2±0.3
lOiO.l
9±0.1
4±0.1
13±0.1
5±0.1
6±0.1
3±0.2
18±0.2
0.5
1.7±.4
0
2.3±0.4
8±0.2
7±0.2
1±0.1
10±0.2
3±0.2
3±0.2
2±0.1
I4±0.1
Bacills substitus
1
4.2±0.5
0
4.3±0.5
13±0.2
8±0.1
4±0.2
11±0.2
4±0.1
4±0.2
5±0.1
16±0.2
0.5
1.4±0.2
0
2.4±0.3
lOiO.l
5±0.2
2±0.2
8±0.2
3±0.2
2±0.1
3±0.2
12±0.2
Psedomonas aeruginose
1
0
0
5.2±0.6
8±0.1
6±0.1
3±0.2
lOiO.l
6±0.1
5±0.1
4±0.2
16±0.2
0.5
0
0
2.4±0.5
6±0.2
4±0.2
0.2±00.1
7±0.2
3±0.2
3±0.2
2±0.1
13±0.2
E.c-Escherichia coli, S.A-Staphylococcus aureus, F.a-Pseudomonas aeruginosa, B.s- Bacillus subtilius, S.t-Salmonella typhi, P.s-Psedomonas syringae, K.p-Klebsiella pneumonia
Table 7.2.9
Antifungal activity data of compounds 19(a-c) and 22 (a-g)
Comp
19a
19b
19c
22a
22b
22c
22d
22e
22f
22g
Fluconazole
Aspergillus flavas
1
4±0.3
0
5.2±0.3
9±0.2
4±0.2
6±0.2
6±0.1
0
5±0.1
7±0.2
14±0.2
0.5
3±0.5
0
2.3±0.4
6±0.2
2±0.2
4±0.4
5±0.2
0
3±0.2
5±0.3
lOiO.l
chrysosporium
keratinophilimi
1
4±0.5
0
4.3±0.5
8±0.2
4±0.l
5±0.2
7±0.2
0
3±0.1
5±0.1
16±0.2
0.5
2.5±0.2
0
2.4±0.3
7±0.2
1±0.1
3±0.2
6±0.1
0
2±0.1
2±0.2
14±0.2
Candida albinus
1
0
0
5.2±0.6
10±0.2
5±0.1
6±0.1
8±0.2
0
4±0.2
6±0.2
23±0.2
0.5
0
0
2.4±0.5
7±0..1
2±0.2
4±0.2
7±0.2
0
1±0.1
4±0.1
20±0.2
198
CHapter 7 'Evatuation of (BiobgicaC activities
7.2.2. Results and discussion
The results of zone of inhibition of antibacterial and antifungal activity of
synthesized compounds 29(a-o) are tabulated in Table 7.2.1. Among the tested
compoimds, 29b, 29h and 29i have shown excellent antibacterial activity against all
the tested bacterial strains. Compoimds 29n and 29o showed very good antibacterial
activity against Salmonella typhi and Salmonella paratyphi. The compounds 29a and
29d have shown equipotent antibacterial activity against Staphylococcus aureus.
Antifungal activity results indicated that compounds 29a and 29b were active against
Microspora griseous. Compoxmds 29c and 29e showed very good antifimgal activity
against C. albicans and compounds 29e, 29(g-o) were inactive against Microspora
griseous.
The antimicrobial activities of test compoimds (39a-m) were compared with
standard marketed drug ampicillin for bacteria and flucanozole for fungi species
respectively. The activities of the compounds were carried out at two different
concentrations (50 and 100 |xg/ml). From the Table 7.2.2, it is evident that the
Compounds 39a, 39e, 39f, 39g, 391 and 39iii have shown good activity against all
tested organisms at both concentrations whereas compound 39j has good activity
against only fungal species Aspergillus Niger and Trichoderma virdae at both
concentrations. The results revealed that on varying substitution on phenyl ring of the
parent molecule 6-phenyl[l,2,4]triazolo[l,5-fl]pyridine, varying degree of activities
were found against different species of tested organisms. Compoimds with -CHO
group on meta position (39a), CI on meta, F on para position (39e), N02 group on
para position (39f), CI on both meta and para position (39g) have showed good
activity against all the species revealing that the halogen substitution enhances the
activity. Replacing 6- phe nyWng of 6-phenyl [1,2,4] triazolo[l,5-a]pyridine with
199
CHapter 7 'Evaluation of(BiobgicaCactivities
pyridine ring (39k) showed decrease in activity against all the species but when
substituted with indole group (39m) retained activity. The position of aldehyde group
from Meta to para position led to decrease in activity.
The results revealed that compounds showed varying degrees of inhibition
against the tested microorganisms. Compounds 30a, 30d, 30e, 32a, 32b, 32d, 32h and
32f showed good activity against Gram-positive bacteria S. aureus (inhibition zone >
17 mm), while compoxmds 30b and 30c were exhibited moderate activity (inhibition
zone 11 to 16 mm). The compounds 32a-b, 32d and 32h exhibited excellent activity
against K. pneumoniae, whereas other compounds were moderately active.
Compounds 30a, 30c, 30d, 32a, 32b, 32f and 32h were moderately active against P.
aeruginosa, compounds 32a, 32b and 32d showed inhibition against S. typhi, while
other compounds showed moderate action. Compound 32f showed comparatively
better activity against S. paratyphi, while the other compounds were either weakly
active or completely inactive (inhibition zone <10 mm).The inhibitory activity against
the tested Gram-negative bacteria E. coli was significantly lower than the other tested
microorganisms. Only compoimd 32a was significantly active, while other
compoxmds were either moderate in their action or completely inactive (Table 7.2.3).
The synthesized compounds were also screened for their in vitro antifungal
activity. The primary screening was carried out using agar well diffusion method [31]
against six fungal strains Aspergellus flavous, Candida albicans, Microspora
griseous, Aspergellus terus and Chrysosporium keratinophilum and the results are
given in Table 7.2.4. The compounds 30e, 32e and 32f have shown promising
antifungal activity against all the tested fungal organisms. Compoimd 30a displayed
better activity against Microspora griseous and Chrysosporium keratinophilum
organisms. Compoimd 30d showed very good activity against Microspora griseous
200
chapter? T.vaCuation of(BioCogicaCactivities
and Aspergellus terus whereas the compound 32b displayed moderate to good activity
against Aspergellus flavous, and Microspora griseous. Compound 32g displayed
moderate to good activity against Aspergellus flavous; Microspora griseous and
Aspergellus terus whereas compound 32h is active against Candida albicans,
Microspora griseous and Aspergellus terus.
Furthermore, the compounds which showed good zone of inhibition were
studied for minimum inhibitory concentration (MIC) using the micro-dilution
susceptibility method in Luria- Bertini agar medium [32] to quantify the antimicrobial
potency of the compounds.
The results given in Table 7.2.5 revealed that, the compoimds exhibited
variable minimum inhibitory concentration (MIC). Compounds 30a-c exhibited very
good antibacterial activity with MIC value 25.9-30.8 M against S. aureus, compounds
30d showed excellent antibacterial activity with MIC ranging between 17.8-23.8 M
against P. aeruginosa and S. aureus whereas compound 32h exhibited promising
activity with MIC 16.8 and 14.4M against S. aureus and S. typhi respectively.
The compoimd 30a exhibited excellent antifungal activity with MIC 18.5M against
Aspergellus flavous and it is inactive against other tested fungal strains. The
compound 30b, showed good antifungal activity with MIC 26.4M against A. flavous,
and A.terus. Whereas compounds 30d and 32f showed moderate to good fimgal
inhibition with MIC values ranging between 23.8M against A. flavous. Compoimds
32a showed good activity with MIC value 20. IM against A. flavous, compound 32d
exhibited excellent activity against C.albicans with MIC 15.3M and the compound
32h substituted with two CF3 group displayed very good activity with MIC 19.2 and
21.6 against A.terus aoA C. keratinophilum respectively.
201
CHapter 7 'EvaCuation of (BiobgicaC activities
Among the imidazo[2,l-6][l,3]thiazole (30a-e) and imidazo[2,l-6] [1,3]
benzothiazole (32a-h) derivatives, it was noticed that the substituents at position -3
and -4 has great influence on the antibacterial activity [33]. It also seems that the
presence of fluoro, chloro, cyano or nitro functional groups enhance the activity of the
parent moiety.
The newly synthesized compounds (29a-o) were screened for their
antibacterial activity against Staphylococcus aureus, Klebsiella pneumonia. S.t-
Salmonella typhi, Escherichia coli, Pseudomonas aeruginosa, and Salmonella
paratyphi. The preliminary test was done using agar-well diffusion method at 1.64,
0.64, and 0.25 mg/mL concentration. The Diclofenac was used as standard drug for
antibacterial from the Table 7.2.6 the investigation of antibacterial screening revealed
that, test compounds showed varying degree of activity against all the tested
microorganisms. Among the tested compounds, compoimd 29c and 29e were foimd to
be most active against all the tested strains and the compoimds 29g and 29h showed
very good activity against all the tested strains.
The newly synthesized compounds (29a-o) were also screened for antifungal
activity. Table 7.2.7 displayed the antifungal activity data of compounds (29a-o)
against, Microspora griseous and Candida albicans. The Fluconazole was used as
standard drug for antifungal activity. The investigation of antifungal screening
revealed that, the compouds 29c exhibited very good antifungal activity against both
the tested fungal strains and the compoimd 29e showed moderate to good activity
against Candida albicans. Compounds (29g-i) and (29m-o) were inactive against
Microspora griseous. The activity of these compounds was foimd to be concentration
dependent.
202
CHapter 7 'EvaCuation of (BiobgicaC activities
The investigation of antibacterial screening revealed that the test compounds
showed varying degree of activity against all the tested microorganisms. Among the
tested compounds 22a and 22d displayed very good activity with zone of inhibition 6-
13mm. compoimd 19b is inactive against all the tested bacterial strains. The rest of
the compounds displayed less activity (Table 7.2.8).
The results given in Table 7.2.9 showed antifungal activity of synthesized
compounds 19(a-c) and 22(a-g). From the table it revealed that, the compounds 22a
showed good antifungal activity against all the fungal strains. Compoimd 22g
displayed good activity against Aspergillus flavas. Compounds 19b and 22g are
inactive against the tested fungal strains.
7.3. Antioxidant activity
Human bodies are protected from oxidative stress by natural enzymatic and
non-enzymatic antioxidant defensive system, whose capacity is affected by age, diet,
and health status of the individual [41]. Therefore, only endogenous antioxidant
defenses are not absolutely efficient. Dietary antioxidants are required to diminish the
cumulative effects of oxidative damage due to excess ROS (reactive oxygen species)
that remains in our system [42]. These free radicals may oxidize nucleic acids,
proteins or lipids which occurs through a chain reaction and may form potentially
toxic end products [43, 44]. Therefore, oxidation can cause not only deterioration of
food stuffs but also harm living organisms. Free radicals are responsible for large
number of diseases including cancer [45], cardiovascular diseases [46], neural
disorders [47], Alzheimer's disease [48], alcohol-induced liver disease [49], early
aging and diabetes [50]. Therefore, the search for new antioxidants has received much
attention.
203
CHapter 7 'EvaCuation of (BiohgicaC activities
The main characteristic of an antioxidant is its abihty to entrap free radicals.
Highly reactive free radicals and oxygen species are present in biological systems
from a wide variety of sources. These free radicals may oxidize nucleic acids,
proteins, lipids or DNA and can initiate degenerative disease [51, 52].
The evaluation of antioxidant activity was done by using following three methods
Free radical scavenging activity by DPPH metliod
DPPH test which is based on the ability of DPPH as a stable free radical to
decolorise in the presence of antioxidant is a direct and reliable method for
determining radical scavenging action [53]. Therefore, the DPPH radical scavenging
activity was determined by the decrease in absorbance at 517 nm due to the reduction
by the antioxidant (AH) or the reaction with a radical species as shown in the eq.l
[54].
DPPH' + R" ^ DPPH-R I
Method of testing
Free radical scavenging activity by DPPH method
Free radical-scavenging capacities of synthesized compounds were determined
according to the reported procedure [55], The newly synthesized compounds at
different concentrations (25-100 jig/mL) were added to each test tube and volume was
made up to 4 ml using methanol. To this 3 mL of 0.004% DPPH in methanol was
added and the mixtures were incubated at room temperature under dark condition for
30 min. The absorbance was recorded at 517 nm using UV-Visible spectrophotometer
(Shimadzu UV-1800, Japan). Butylatedhydroxytoluene (BHT), dissolved in distilled
water was used as a reference. Control sample was prepared using the same volume
without any compoxmd and BHT, 95% methanol served as blank. Test was performed
204
Cfiapter 7 'EvaCuation of (BiobgicaC activities ! •
in triplicate and the results were averaged. Free radical scavenging activity was
calculated using the formula:
% of radical scavenging activity = [(Acomroi- Aiest)/Acontroi] >< 100
Where Acontroi is the absorbance of the control sample (DPPH solution without test
sample) and Atest is the absorbance of the test sample (DPPH solution + test
compoxmd).
Table 7.3.1
Scavenging effect of selected synthesized compounds on stable radical 1, 1-diphenyl-
picrylhydrazyl (DPPH) 30(a-e) and 32(a-h)
Compounds
30a
30c
30d
30e
32a
32b
32d
32f
32h
BHT
DPPH Assay in %
64.6±0.630
18.1±0.169
25.36±0.402
24.78±0.730
65.2±0.260
15.3±0.251
59.3±0.864
27.3±0.325
58.2±0.189
90.01±0.469
Each value is expressed as meaniSD of three replicates Butylatedhydroxytoluene (BHT) used as standard
205
CRapter? 'Evaluation of <BiolJogicaC activities
Table 7.3.2 DPPH assay in % of synthesized compounds 29a-o
Comp 29a 29b 29c 29d 29e 29f 29g 29h
DPPH Assay in % 52.5 ±0.45 37.6±0.61 26.3 ±0.23 43.3±0.16 38.7±0.35 67.3±0.12 32.5 ±0.45 54.1±0.14
Comp 29i 29j 29k 291
29m 29ii 29o
BHT
DPPH Assay in % 22.5 ±0.45 37.2±0.26 28.7±0.11 26.3 ±0.23 38.6±0.61 56.2±0.35 78.3±0.16 90.42±0.25
Table 7.3.3
Scavenging effect of selected synthesized compounds on stable radical I, 1-diphenyl-
picrylhydrazyl (DPPH) 19(a-c) and 22 (a-g)
Comp
22a
22b
22c
22d
22e
22f
22g
BHT
DPPH Assay in %
64.6
32.5
39.7
62.3
16.1
22.3
58.7
94.3
206
CHapter 7 Tivafuation of (BiobgicaC activities
Results and discussion
The investigation of (DPPH) radical scavenging activity (Table 4) revealed
that, among the tested compounds, compounds 3a, 5a, 5d and 5h which contains
electron withdrawing group(s) on phenyl ring have shown promising antioxidant
property when compared to standard. The incorporation of electron donating group
CH3 (5f) on target compounds decreases the antioxidant property. The remaining
compoimds 3c, 3d, 3e and 5b displayed less activity (Table 7.3.1).
The investigation of (DPPH) radical scavenging activity (Table 7.3.2)
revealed that, among the tested compoimds, compounds 4f, and 4o which contains
electron withdrawing group(s) on phenyl ring have shown promising antioxidant
property when compared to standard. The incorporation of electron donating group
CH3 (4h) on target compounds decreases the antioxidant property. Compounds 4a and
4n displayed moderate to good activity. The remaining compounds displayed less
activity.
The investigation of (DPPH) radical scavenging activity (Table 7.3.3) of the newly
synthesized compounds 19(a-c) and 22 (a-g) revealed that, compounds 22a, 22d
which contains electron withdrawing group(s) on phenyl ring have shown promising
antioxidant property when compared to standard. The remaining compounds
displayed less activity.
207
Cfiapter 7 'Evaluation of (BiobgicaC activities
References
[I] Shankar GA, Kallanagouda RA, Eur. J. Chem. 2011, 2,94.
[2] Nuray UG, Omer K, Eur. J. Med. Chem. 2010, 45, 63-68.
[3] Andreani A, Rambaldi M, Mascellani G, Rugarli P, Eur. J. Med. Chem.
1987, 22, 22.
[4] Andreani A, Bumelli S, Granaiola M, Leoni A, Locatelli A, Morigi R,
Rambaldi M, Varoli L, Calonghi N, Cappadone C, Famiggia G, Zini M,
Stefanelli C, Masotti L, Radin NS, Shoemake RH, J. Med. Chem. 2008, 5,
1816.
[5] Elshorbagi AN, Sakai S, Elgendy MA, Omar N, Faraq HH, Chem. Pharm.
Bull. 1989, 37, 2975.
[6] Mase T, Arima H, Tomioka K, Murae K, J. Med. Chem. 1986, 29, 394.
[7] Mase T, Arima H, Tomioka K, Yamada T, Murase K, Eur. J. Med. Chem.
1988, 23, 339.
[8] Narayana B, Raj KKV, Ashalatha BV, Kumari NS, Phosp.Sul Silt. Rltd.
Elem., 2007, 182, 14.
[9] Vinayak SH, Kolavi GD, Ravi SL, Khazi lAM, /. Sul. Chem. 2006, 27, 569.
[10] Venkatesh KB, Yadav DB, Curr. Chem. Bio. 2010, 4, 150.
[II] Nardkami AB, Kamath RV, Khadse GB, Ind. J. Meter. Chem. 2000, 9, 310.
[12] Tanabe Y, Kawai A, Yoshida Y, Ogura M, Okumura H, Heterocycles, 1997,
45, 1588.
[13] Trapani G, Franco M, Lofta A, Reho A, Liso G, Eur. J. Pharm. Sci. 2001,
14, 216.
[14] Bhaskary V.H., Puli K., Sangameswaran B., Jayakar B., Ind. J. Meter. Chem.
2006, 5, 300.
208
Cdapter 7 'EvaCuation of (BiobgicaC activities
[15] Hozien Z.A., El-Wareth A.O.S.A., El-Sherief H.A.H, Mahmoud A.M., J.
Meter. Chem. 2000, 37, 949.
[16] Andreani E., Eur. J. Med. Chem. 2001, 36, 746.
[17] Maglich J.M., Parks D.J., Moore L.B., Collins J.L., Goodwin B., Billin A.N.,
Stoltz C.A., Kliewer S.A., Lambert M.H., Willson T.M., Moore J.T., J. Biol.
Chem. 2003, 278, 17283.
[18] Chua T., Moore C.L., Perri M.B., Donabedian S.M., Masch W., Vager D.,
Davis S.LLulek., K., Zimnicki B., Zervos M.J., J. Clin. Microbiol. 2008, 46,
2345.
[19] Chu D.T.W., Plattner J.J., Katz L., J. Med. Chem. 1996, 39, 3853.
[20] (a) Shapiro, S. L.; Soloway, H.; Freedman, L. J .Am. Pharm. Assoc. Sci. Ed.
1957, 46, 333. (b) Houlian, W. J. U. S. Patent, 3526626, 1970. (c)
Bender, P. E.;Hanna,N. U.S. Patent, 4719218,1988.
[21] Rupert, K. C ; Henry, J. R.; Dodd, J. H.; Wadsworth, S. A.; Cavender, D.
E.; Olini, G. C ; Fahmy, B.; Siekierka, J. J. Bioorg. Med. Chem. Lett. 2003,
13, 347.
[22] C6cile E. Jean-Louis R. Hassan, A. Jean-Michel, Eand Alain, G, Chem.
Pharm. Bull. 2000, 48, 940.
[23] Rival Y, Grassy G, Michel G. Chem. Pharm. Bull.1992, 40, 1170.
[24] (a) Fisher MH, Lusi A. J. Med Chem. 1972, 15, 982. (b) Rival Y Grassy G,
Taudou A, Ecalle R. Eur. J. Med Chem. 1991, 26, 13.
[25] (a) Hamdouchi C, de Bias J, del Prado M, Gruber J, Heinz BA, Vance L. J.
Med. Chem. 1999, 42, 50. (b) Lhassani M, Chavignon O, Chezal JM,
Teulade JC, Chapat JP, Snoeck R, Andrei G, Balzarini J, De Clercq E,
Gueiffier A. Eur. J. Med. Chem. 1999, 34, 271.
209
Cfiapter 7 'Evafuation of (BiobgicaC activities • l^BI
[26] Venkatesh KB, Yadav. D. B. Curr. Chem. Biol. 2010, 4, 150.
[27] Suman S, Trissa J, Halligudi SB, J Molec. Cata. A: Chemical 2006, 244,
182.
[28] Sriram D, Banerjee D, Yogeeswari P. J. Enzyme Inhib. Med. Chem. 2009,
24,5.
[29] Ki-Whan C, Yoon S, Kwang TS, Tae Ho P, Jeong Soo A. Bull. Korean
Chem. Soc. 1999, 20, 8973.
[30] Hemnann DJ, Peppard WJ, Ledeboer NA, Theesfeld ML, Weigelt JA,
Buechel B. J. Expert Review of Anti-infective Therapy. 2008, 6, 848.
[31] Vidya SM, Krishna V, Manjunatha BK, Rajesh KP, Bharath, Manjunatha
H. DOI 10:1007/s00044-011-9614-4, A/e^C/ie/w;?e5, 03. 03.2011.
[32] Dick JD, Merz W.G, Saral R. Antimicro. Agents Chemo, 1980, 18, 158.
[33] Ananthnarayan and Panka. ATB of Medical Microbiology, Himalayan
Publications, 7th edition, (2004).
[34] Ryley J.F, Wilson R.G, Baratt-Bee K.J. Sabouraudia, 1984, 22, 53.
[35] Stiller R.L, Bennett J.E, Scholer H.J, Wall M, Polok A, Stevens D.A, J.
Infect. Dis, 1983, 147, 1070.
[36] White T.C, Marr K.A, Bowden R.A. Clin. Microbiol. Rev, 1998, 11, 382.
[37] Runyoro K.B.D, Matee I.M, Ngassapa D.O, Joseph C.C, Mbwambo Z.H.
BMC Complement Altern Med, 2006, 6, 11.
[38] Ilangovan A, Venkatesan P, Sundararaman M, Rajesh Kumar R. Med Chem
Res,mi,2\,69A.
[39] Nasser khalil S.A.M. Eur J Med Chem., 2010, 45, 5265.
[40] National committee for clinical laboratory standards (NCCLS) (1982)
Standard methods for dilution antimicrobial susceptibility tests for bacteria,
210
CHapter 7 'EvaCuation of <Bio(bgicaC activities
which grows aerobically; Villanova. 242.
[41] Chun 0.K, Kim D.O, Lee C.Y. J. Agric. Food. Chem. 2003, 51, 8067.
[42] Lim Y.Y, Murtijaya J. Food. Sci. Technol, imi, 40, 1664.
[43] Guo J.N, Weng X.C, Wu H, Li Q.H, Bi K.S. Food.Chem. 2005, 91, 287.
[44] Chunhuan H, Xiao wen J, Yingming P, Hengshan W, Kai W, Min L, Lizhu
Y. Med.Chem. Res. 2010, 9, 448.
[45] Kinnula V. L, Crapo J. D. Free. Radio. Biol. Med, 2004, 36, 718.
[46] Singh U, Jialal L Pathophysiology 2006, 13, 129.
[47] Sas K, Robotka H, Toldi J, Vecsei L. J. Neurol. Sci, 2007, 257, 221.
[48] Smith M.A, Rottkamp C.A, Nxmomura A, Raina A.K, Perry G.
Biochim.Biophys. Acta. 2000, 1502,139.
[49] Arteel, A. Antonello, A.Tarozzi, F. Morroni, A. CavalU, M. Rosini, P.
Hrelia, M.L. Bolognesi, C. Melchiorre. J. Med. Chem, 2006, 49, 6642.
[50] Hyun D. H, Hernandez J. O, Mattson M. P, de Cabo R. A^ng. Res. Rev,
2006, 5, 209.
[51] Jung H.A, Park J.C, Chung H.Y, Kim J, Choi J. S. Arch. Pharm. Res, 1999,
22,213.
[52] Pietta P, Simonetti P, Mauri P. J. Agric. Food. Chem, 1998, 6, 4487.
[53] Niki E. Chem. Phys. Lipids, 1987, 44, 227.
[54] Gordon M.H, Pokomy J, Yanishlieva N, Gordon M. H. (Eds.), Antioxidants
in Food: Practical Applications. Wood head Publishing Limited,
Cambridge. (2001) 7.
[55] Satheeshkumar D, Muthu K.A, Manavalan R. J. Pharm. Res, 2011, 4, 976.
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