Shaikh Nuzhat Begum. et al. / Journal of Pharmaceutical Biology, 4(1), 2014, 4-8.
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EVALUATION OF ANTIBACTERIAL ACTIVITIES OF SOME
HALOGEN SUBSTITUTED HYDROXYFLAVONES
Shaikh Nuzhat Begum1, Shivaji B. Chavan2, Mohammad A. Baseer2, Vaseem Baig MM1*
1
PG Department of Biotechnology, 2Organic Chemistry Research Laboratory,
Yeshwant Mahavidyalaya, Nanded, 431602, Maharashtra, India.
ABSTRACT
A new series of halogen substituted flavones (a-p) were tested for their in vitro antibacterial activity against four
different pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Corynebacterium sp and Xanthomonas
malvacerum using Streptomycin as standard drug. The results reflect promising moderate to good activity at different
dilutions against different strains of bacteria employed.
Keywords: Halohydroxy chalcones, Flavones, Antibacterial activity.
INTRODUCTION
Flavones constitute one of the major class of
naturally occurring compounds. Synthesis and
antibacterial, antifungal activity studies of Flavones and
their derivatives have attracted considerable attention due
to their significant biological activities [1-3]. Many
substituted flavonoids exhibit a broad range of
pharmacological activities such as Analgesic [4], Antiinflammatory [5], Chemopreventive [6], Anti-cancer [7,
8], Antitumor [9], Antioxidant [10], Antiglycation [11],
Vasorelaxant [12], Cyclooxygenase-2 (COX2) [13],
Antiplaque [14], Anti-tuberculosis [15]. It is well
documented that, halogen substituted flavonoid
compounds are also strongly biologically active [16] but
to our knowledge very few synthetic flavonoids have been
reported with halogens as substituents. On the other hand,
halogen and methoxy group substitution in pyron ring
also exhibit the biological activity [17]. There has been a
particular interest in the synthesis of halogen substituted
flavones. In view of these observations, in the present
investigation, we report herein the new series of flavones
(a-p), having chloro, bromo, iodo and methoxy moieties
in pyron ring with an aim to find new most active
antibacterial agents. All the compounds were tested for
their in vitro antibacterial activity against Escherichia
coli, Staphylococcus aureus, Corynebacterium sp. and
Xanthomonas malvacerum using Streptomycin as
standard drug.
MATERIALS AND METHODS
Cultures
The Cultures of the bacteria used Escherichia
coli and Staphylococcus aureus were procured from
Department of Microbiology, Dr. Shankarrao Chavan
Govt. Medical College, Nanded. While culture of
Corynebacterium sp. was obtained from National
Chemical Laboratory, Pune.
The bacterial culture of Xanthomonas
malvacerum was available at P. G. Department of
Biotechnology, Yeshwant College, Nanded.
The cultures were maintained on the medium
suggested by the respective laboratory and sub-culturing
was done fortnightly. The cultures were incubated in an
incubator, while the cultures were stored in refrigerator.
Sr.No
1
2
3
4
Organism
Escherichia coli
Staphylococcus aureus
Corynebacterium sp.
Xanthomonas malvacerum
Culture Medium
Nutrient Agar (NA)
Nutrient Agar
Nutrient Agar
Nutrient Agar
Media
The following media were used in this study;
Nutrient Agar
Beef extract (1gm); Yeast extract
Corresponding Author:- MM Vaseem Baig Email:- [email protected]
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(2gm); Peptone, bacteriological (5gm);Sodium Chloride
(5gm); Agar (15gm); Distilled water (1000ml); Ph (7.4).
The media were sterilized at pressure 15 lb. for 20
minutes in an Autoclave. 8ml of medium was poured in
each test tube for slant preparation and 25ml of liquid
media was poured in each 250ml conical flask for enzyme
production.
Culture Vessels
All the glasswares were thoroughly cleaned with
acid-dichromate mixture and then washed with hot water
and rinsed with distilled water and dried completely
before use.
Inoculum
Spore/cell suspension was prepared by adding
sterile water 48 hr old for bacterial NA slant culture and
0.5ml of it was used as inoculum in all experiments. In
every case bacterial suspension was standardized to
7
contain 10 cells/ml by using haemocytometer. All the
treatments /samplings were done in triplicates and results
have been presented after repeating the experiment.
Incubation
Cultures were incubated at 27 ± 2ºC in the
laboratory and as far as possible the temperature was
maintained constant during the incubation and course of
study. In all experiments the cultures were incubated for
2 days.
Preparation of Compound Solutions
The flavonoid compounds collected were
purified by recrystallisation or column chromatography
using suitable solvents like ethanol/ methanol or acetoneethyl acetate. The pure, dry compounds are then weighed
and dissolved in DMSO. These solutions are used to
assess their antibacterial potential.
Flavone solutions
1 mg of the compound was dissolved in 1-2
drops of DMSO, then volume was made to 1 ml with
sterile distilled water. Thus by making the concentration
of the solution as 1000 µg/ml.
Flavonoids for Antibacterial Properties
Antibacterial activities of the flavonoids were
evaluated by Well-diffusion method expressed by zone of
inhibition mm in diameter for bacterial test organisms.
The bioassay was carried out by using 1ml of
inoculum (107 colony forming units) prepared from an
overnight Nutrient Broth culture for given test bacteria
were used. 1ml of the resultant cell suspension was
poured in the Petri plate and the plates were poured with
respective medium was used to seed each prepared plate.
The medium were allowed to solidify. Using a sterilized
cork borer wells of 5mm diameter were made in the
solidified inoculated medium and the plate area
uniformly. The wells were filled with 0.5ml of compound
solution. Plates were then incubated aerobically at 37ºC
for 24 h for bacteria.
Similarly,
wells
containing
standard
concentration of antibiotics (Streptomycin ) was used to
compare the antibacterial property of the flavonoid. Pure
antibiotics (Streptomycin) were purchased from Himedia, Mumbai. One hundred mg of each antibiotic was
dissolved separately in 1000 ml sterile distilled water;
0.5ml was used to fill the wells. These pure antibiotic
solutions were used for comparison.
Antibacterial Activity of Flavones
The experiments were performed to check the
antibacterial properties of flavones (a-p) against selected
bacterial pathogens. The compounds were dissolved in
Dimethyl Sulphoxide DMSO as explained in material and
methods and accordingly these dilutions were made; the
activity was done by Well method[18]. The zone of
inhibitions in mm recorded for different flavones against
the bacteria are tabulated as Table 1.
RESULTS AND DISCUSSION
The results for the general screening for
antibacterial activity exhibited by the synthetic flavonoids
were studied. In the screening, it was clear that most of
the flavonoids possess antibacterial activity with few
exceptions.
It was clear that, polyhydroxy flavones exhibited
maximum activity against the selected test bacteria. The
effect of synthetic flavonoids like halogeno hydroxyl
flavones for antibacterial activities against selected human
and plant bacterial pathogens in vitro was undertaken.
The study was limited to sixteen halogeno hydroxy
flavones with substituent groups as mentioned. The
activity of flavones with less number of –OH groups were
relatively less than the activity of flavones with more
number of –OH groups. Similarly, the activity of flavones
was comparatively less for nonhalogen substituted
hydroxyl flavones than the halogen substituted hydroxy
flavones.
Greater and remarkable antibacterial activities of
the flavones tested were recorded with Staphylococcus
aureus and Corynebacterium sp. whereas E. coli and
Xanthomonas malvacearum were comparatively less
inhibited. These flavones showed better activity against
the Gram-positive than the Gram-negative bacteria. The
results support the observation that Gram-negative
bacteria are more resistant, probably because of their
thick murein layer which prevents the entry of inhibitors.
The flavones with more number of –OH groups
inhibited both Gram-positive bacteria, and the Gramnegative, thereby suggesting a broad spectrum
antibacterial property of these compounds like Compound
‘p’. This seems to justify their usage for the treatment of
infections by the indigenous people of this region. The
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concentration of the flavonoids 50 µg/ml, 100µg/ml,
200µg/ml, 300µg/ml and 500µg/ml tested in the
experiment was much similar to the pure antibiotic with
the same concentrations. This indicates that the flavone
‘p’ used in the experiments contains antibacterial
component similar to the pure antibiotic.
Though the flavones tested produced varied
inhibition zones, the highest inhibition zone of 30 mm
was produced by the flavone ‘p’ followed by flavone ‘f’
flavone ‘l’ (29 mm) against Staphylococcus aureus . The
antibacterial activity of these flavones was approximately
equivalent to 500µg/ml of Streptomycin whereas
compounds c, g, h, k, m and ‘o’ exhibited good activity
against Staphylococcus aureus. The compounds ‘o’ and
‘p’ are moderately active against Corynebacterium sp.
All the flavones are biologically active against E. coli
pathogen but the highest activity is shown by flavone ‘p’
as mentioned in the Table 1. The compounds ‘l’ and ‘p’
showed maximum activity against Xanthomonas
malvacearum while c, k, m and ‘o’ showed moderate
activity in comparison with standard drug. The
brominated compounds in general with more number of
hydroxyl groups exhibited most antibacterial activity
against tested pathogens as comparison with
Streptomycin. The presence of flavone moiety, the
substituents particularly bromo, chloro, iodo and methoxy
groups in phenyl ring may be responsible for
antimicrobial activity of this class of compounds.
Figure 1. Description of Flavones
R5
R6
R1
R2
O
R3
R7
R8
R4
O
R8= H
Table 1. Effect of Flavones on Growth of different Bacteria
S. aurius
E. coli
Zone of inhibition in mm
Zone of inhibition in mm
for concentration (µg/mL)
for concentration (µg/mL)
Compd.
10 20 30 50
10 20 30 50
50
50
No.
0
0
0
0
0
0
0
0
05 10 12 16 24 05 10 12 16 20
a.
06 10 12 18 24 04 09 12 15 20
b.
08 12 14 18 28 05 09 12 16 22
c.
06 09 14 17 25 04 06 11 13 19
d.
04 08 10 15 24 02 05 08 12 18
e.
06 14 14 18 29 04 10 12 14 19
f.
05 13 14 18 27 03 09 10 13 20
g.
07 12 12 17 28 05 10 11 15 21
h.
06 10 11 18 26 04 08 10 14 20
i.
05 11 12 18 26 03 07 09 12 18
j
06 12 14 18 28 04 08 10 14 22
k.
05 12 13 18 29 05 10 13 14 22
l.
05 13 14 20 28 05 10 12 13 21
m
04 10 12 17 25 04 08 10 12 20
n.
06 12 14 18 28 06 10 11 14 22
o.
07 14 15 22 30 07 11 13 15 24
p.
Streptomycin 08 13 15 20 31 06 10 13 16 26
X. malvacerum
Zone of inhibition in mm
for concentration (µg/mL)
10 20 30 50
50
0
0
0
0
03 07 08 12 17
03 07 08 11 18
04 08 09 13 19
04 08 07 11 17
02 05 06 10 15
05 06 06 11 17
04 07 08 11 17
05 08 07 12 18
04 08 08 11 16
04 06 06 10 16
05 07 08 10 18
04 08 08 12 19
05 07 09 12 18
05 06 08 11 17
06 07 08 10 18
06 08 10 12 20
06 09 11 14 22
Corynebacterium sp.
Zone of inhibition in mm for
concentration (µg/mL)
10 20 30
50
500
0
0
0
04 09 12 15
20
03 09 13 16
20
06 12 15 18
23
05 10 14 16
21
03 09 12 15
19
06 11 14 17
21
05 10 13 16
20
06 12 15 17
22
05 10 13 17
21
04 09 12 15
19
06 12 15 19
24
05 12 15 18
23
06 11 14 17
22
06 12 15 17
21
08 13 17 21
25
07 13 17 21
26
08 14 18 23
32
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Compd. No.
a.
b.
c.
d.
e.
f.
g.
h.
i.
j
k.
l.
m
n.
o.
p.
R1
OH
Br
Br
Br
I
OH
I
Br
Br
I
OH
Br
Br
I
OH
Br
R2
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
OH
OH
R3
Br
OH
OH
Br
I
Br
OH
OH
Br
I
Br
Br
OH
OH
Br
Br
CONCLUSION
In summary, all the newly synthesized halogen
substituted flavone compounds (a-p) were checked for
their antibacterial activity. It is concluded from the results
that, Cl, Br, I and methoxy substituents on the flavones
ring are responsible for the enhancement of the
antibacterial activity. It can further be concluded that the
inhibition increases with increasing the hydroxyl groups
and halogen atoms on the flavones ring. From the results
it is evident that most of chloro, bromo, iodo and
methoxy group substituted compounds exhibited
good
R4
H
H
OH
H
H
H
H
H
H
H
H
H
H
H
H
OH
R5
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
OH
OH
OH
OH
OH
R6
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OH
OH
OH
OH
OH
R7
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
antimicrobial activity. There is much scope to synthesise
and screen further new halogeno hydroxy flavones against
a large number of gram positive and gram negative plant
and human pathogens in order to find better antimicrobial
agents.
ACKOWLEDGEMENT
The authors are thankful to the Principal,
Yeshwant College, Nanded for providing necessary
facilities for carrying out the research work.
REFERENCES
1. Rao KV, Chattopadhyay SK, Reddy GC, Flavonoids with mosquito larval toxicity, J. Agric. Food Chem, 38, 1990,
1427-30
2. Weidenborner M, Jha HC. Antifungal Activity of Flavonoids against different fungi occurring on grain. Pestic. Sci., 38,
1993, 347-351
3. Silva AM, Weidenborner M, Cavaleiro JAS. Growth control of different fusarium species by selected flavones. Mycol.
Res., 102, 1998, 638-640.
4. Hasan SM, Alam MM, Asif H, Suruchi K, Mymoona A, Zaman MS. Synthesis of 6-aminomethyl derivativesa of
benzopyran-4-one with dual biological properties: Anti-inflammatory-analgesic and antimicrobial, Eur. J. Med. Chem,
44, 2009, 4896-4903.
5. Dharma TN, Tulasi CP, Madhusudhan RI, Gupa A Koti RV. A facile synthesis of flavones derivatives used as potent
Anti-inflammatory agents. Inter. J. Pharm. Pharm. Sci, 3(2), 2011, 51-54.
6. Robert MM, Simonida G, Bhushan CS, Susantha NC, Om prakash, Rajesh N. Synthesis of Abyssinone II and related
compounds as potential chemopreventive agents. Eur. J. Med. Chem., 41, 2006, 263-267.
7. Jaya P, Bindushree P, Pavankumar K, Rajavel RK, Sivakumar T. Synthesis and investigation of Anti-inflammatory,
Anti-cancer and anti-microbial activity of substituted 3-phenyl Flavones. Inter. J. Pharm. Chem. Res, 1(1), 2012, 28-38.
8. Nicholas JL, David R, Alan T. McGown and John AH. The Total Synthesis of an Aurone Isolated from Uvaria
hamiltonii: Aurones and Flavones as Anticancer Agents. Bioog. Med. Chem. Lett, 13, 2003, 3759-3763.
9. Xin-Hua L, Hui-Feng L, Xu Shen, Bao-An S, Pinaki SB, Hai-Liang Z, Jin-Xing L. Xing-Bao Q. Synthesis and molecular
docking studies of novel 2-chloro-pyridine derivatives containing flavones moieties as potential antitumor agents.
Bioorg. Med. Chem. Lett, 20, 2010, 4163-4167.
10. Elfi Susanti VH, Sabirin M, Tutik DW, Mustofa and Tri Redjeki. Synthesis characterization Antioxidant activity of 7hydroxy-3’, 4’-Dimethoxyflavone. Indo. J. Chem, 12(2), 2012, 146-151.
11. Jayashree BS, Chaturvedi1 P, Venkatachalam H, Chaudary PVR, Yogendra N. Antiglycation and anticancer activity of
some newer synthetic flavones. Der Pharma Chemica, 4(4), 2012, 1626-1630.
7|P a g e
Shaikh Nuzhat Begum. et al. / Journal of Pharmaceutical Biology, 4(1), 2014, 4-8.
12. Xiaowu D, Tao L, Jingying Y, Peng W, Jing C, Yongzhou H. Synthesis, biological evaluation and quantitative structureactivities relationship of flavonoids as vasorelaxant agents. Bioorg. Med. Chem, 17, 2009, 716-726.
13. Thanh-Dao T, Haeil P, Gerhard FE and Khac-Minh T. 2’-Hydroxychalcone Analogues: Synthesis and Structure-PGE2
Inhibitory Activity Relationship. 12th Inter Elec Conf Synth Org Chem, 1-30, 2008, 1-7.
14. Vijayalaxmi CP. Synthesis and invitro antiplaque activity of chalcone, flavones and flavanol derivatives. Inter. J. Pharm.
Sci. Res, 3(12), 2012, 5006-5014.
15. Yuh-Meei L, Yasheen Z, Michael TF, Li-Ming Z, Weiguo N and Fa-Ching C. Chalcones and Flavonoids as AntiTuberculosis Agents. Bioorg. Med. Chem, 10, 2002, 2795-2802.
16. Sohel M, Sayed A and Azizul I. Cytotoxic and antimicrobial activities of some synthetic flavones. Ind. J. Chem, 45B,
2006, 1478-1486.
17. Mokle SS and Vibhute YB. Synthesis of some new biologically active chalcones and flavones. Der Pharma Chemica,
1(2), 2009, 145-152.
18. Barry AL. The Antimicrobial Susceptibility test: Principle and Practice, edited by Illus Lea and Febiger, Philodalphia pa,
(USA), 1976; 180.
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