Download Chapter-6 Biological activity of newly prepared

Chapter-6
Chapter-6
Biological activity of newly prepared compounds
[5a-e to 11a-e]
6.1 Introduction:
In the animal kingdom, including human being, infection is a major category
of disease. A skilled management is crucially important for antimicrobial drugs.
In the past many natural substances were used as therapy. The Greeks used
male fern (Dryopteris filix-mas) as anthelmintics. The Hindus treated leprosy with
chaulmoogra (Hydnocarpus wightiana) and the Africans were using cinchona bark
(Quina) against malaria.
The history of modern chemotherapy did not begin until Paul Ehrlich observed
that aniline dye selectively stained the bacteria. He invented the word ‘chemotherapy’
The term ‘chemotherapy’ means chemical therapy or chemical treatment. It
was first introduced by ‘Paul Ehrlich’.
So he is known as the ‘Father of
Chemotherapy’. Ehrlich defined chemotherapy as the treatment of diseases due to
micro-organisms invades by chemical compound which destroys the micro-organisms
without affecting the tissues of the host.
The ideal requirement of a drug is that it should act at the desired location or a
part of the body when applied. In practice, however, no drug behaves in the said
manner; but it tends to distribute itself almost everywhere in the tissues of the host.
Some species like protozoa, bacteria, viruses, fungi are responsible for several
diseases “Chemical compounds which are used to destroy micro-organism or to
inhibit their growth without injuring any part of body are called – “Chemotherapeutic
Agent”
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Chemotherapeutic Agents which destroy micro-organism is termed as a Cidal
effect while the rest only inhabit growth of the organisms which is termed as Static
effect.
The first compound which was used by Ehrlich was organic dye. Then
onwards organic compounds of diverse chemical structures have been used in
chemotherapy.
A huge amount of different drugs are derived either naturally or synthetically;
and in most of the cases they are heterocyclic compounds. The heterocyclic
compounds like nicotine, morphine and vitamins are well known for their drug
activity. Numbers of nitrogen containing heterocyclic compounds are well known
drugs also used as insecticides, fungicides, bactericides etc. [1-4].
Microbiology is a branch of science that deals with microbes. They are too
small and can be seen only by magnifying them with the help of a microscope.
Microbes are the micro-organisms like viruses, bacteria, algae, fungi, protozoa, etc.
The term Bacteria is the plural form of the Bacterium, which means to be a
single organism. The Dutch scientist Antonie van Leeuwenhoek was the first person
to observe bacteria and other micro-organisms by a single-lens microscope. Apart
from Viruses, Bacteria are the smallest living things on our planet Earth.
Bacteria are found everywhere; in water, in air, in soil and even on plants and
animals (including humans) externally as well as internally. They multiply quite
rapidly and form colonies under favorable conditions. They reproduce by binary
fission. In this process, a single cell divides into two identical daughter cells. Factors
that affect bacteria growth are temperature, pH, humidity and oxygen supply.
Bacteria can survive at a specific ideal temperature and pH range. Most
disease-causing bacteria survive at temperatures close to that of the human body that
is 37°C or 98.6°F; and at pH range of 6.7 to 7.5.
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Few bacteria don’t harm unless they enter the blood. Among these,
Staphylococcus is responsible for the potentially fatal toxic shock syndrome.
Escherichia coli are helpful in the digestive system, but it might cause various
diseases like diarrhea, cramping and possibly even death if it enters into the blood.
Hans Christian Gram, a Danish bacteriologist, discovered a stain known as
Gram stain. With the help of the said, bacteria are divide into two classes - Gram
positive and Gram negative.
For Gram stain; crystal violet is used as a primary stain and safranin is used as
a counter stain. In the said process, if the bacteria turn into purple colour after getting
stained, they are called 'Gram positive’, and they are ‘Gram negative’ if turned into
red colour when counterstained.
A fungus is a member of the group of organisms that includes unicellular or
multicellular microorganisms such as yeasts and molds. They are also responsible for
some diseases.
Bacteria and fungus can damage food or cause diseases. Most forms of food
preservation; such as freezing, heating or drying are designed to kill them or make
them inactivate. One of the most common methods of destroying bacteria in food is
pasteurization. It is this process that the milk can be preserved for quite a long time.
The current challenge for medical science is the resistance power which has
been developed by these disease-causing bacteria against the antibiotics and other
concerned drugs. This evolution makes them able to turn into new forms or secret
certain chemicals to make the antibiotics ineffective.
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6.2
Antibacterial activity:
Following bacteria were selected for in-vitro study.
Bacillus subtilis: Gram positive.
Staphylococcus aureus: Gram positive
Klebsiella pneumoniae: Gram-negative
Escherichia coli: Gram negative
Materials:
All the compounds [5a-e to 11a-e] described in earlier chapters were tested
for their antimicrobial study. All other chemicals used were of laboratory grade.
Various methods have used from time to time by several worker to evaluate
the antimicrobial activity.[5-7] the evaluation can be done by the following methods
[8]
1.
Agar diffusion method.
2.
Agar streak dilution method.
3.
Serial dilution method and
4.
Turbidometric method.
Agar diffusion method is again of three types.

Agar cup method

Paper disc method and

Agar ditch method
In present work, Agar cup method is used.
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Culture medium preparation:
Nutrient agar medium used and Chemical composition of the medium was,
Peptone
1.0 gm
NaCl
0.5 gm
Meat extracts
0.3 gm
Distilled water
100 ml
pH
7.6
Agar
2.0 gm
The ingredient were weighed and dissolved in distilled water, pH was adjusted
to 7.6 and then agar powder was added to it. The medium was dispensed in 25 ml
quantity in different test tubes. The test tubes were plugged by cotton-wool and
sterilized at 121.5°C and 15 pound per square inch (psi) pressure for 15 minutes.
Antibacterial susceptibility testing:
The study has been conducted according to the method adopted by
Cruickshank et al [9] Nutrient agar broth was melted in a water bath and cooked to 45
0C with gentle shaking to bring about uniform cooling. It was inoculated with 0.5-0.6
ml of 24 hour old culture especially and mixed well by gentle shaking before pouring
on the sterilized Petri dish (25 ml each). The poured material was allowed to set (1.5
hour) and there after the “cups” was made by punching into the agar surface with a
sterile cup borer and soaping out the punched part of agar. Into this “cups” test
solution was added by sterile micropipette. The plates were noted.
The antibacterial activities of all compounds are calculated in percentage (%) of
inhibition, shown in Tables: 6.1 to 6.8.
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Chapter-6
The percentage (%) data are shown in all tables as follows;
(+) = Small clearing zone (<50%), slightly active,
(++) = Medium clearing zone (51-55%), moderately active,
(+++) = Large clearing zone (56-60%), highly active and,
(++++) = Very large clearing zone (>60%), very high activity.
Table 6.1: Antibacterial activity of Tetracycline (standard)
% Zone of Inhibition
Sample
Tetracycline
Gram +Ve
Gram –Ve
Bacillus
Staphylococcus
Klebsiella
subtilis
aureus
pneumoniae
++++
+++
++++
E-Coli
++++
Table 6.2: Antibacterial activity of compounds (5a-e)
% Zone of Inhibition
Sample
Gram +Ve
Gram –Ve
Bacillus
Staphylococcus
Klebsiella
E-Coli
subtilis
aureus
pneumoniae
5a
+
+
++++
+++
5b
++
+
++++
+++
5c
++
+
+++
++++
5d
++++
++
++++
++++
5e
++++
++
++++
++++
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Chapter-6
Table 6.3: Antibacterial activity of compounds (6a-e)
% Zone of Inhibition
Sample
Gram +Ve
Gram –Ve
Bacillus
Staphylococcus
Klebsiella
E-Coli
subtilis
aureus
pneumoniae
6a
++
+
++++
++++
6b
++
++
++++
++++
6c
+++
++
+++
++++
6d
++++
+++
++++
++++
6e
++++
+++
++++
++++
Table 6.4: Antibacterial activity of compounds (7a-e)
% Zone of Inhibition
Sample
Gram +Ve
Gram –Ve
Bacillus
Staphylococcus
Klebsiella
E-Coli
subtilis
aureus
pneumoniae
7a
++
+
++++
++++
7b
++
+
++++
+++
7c
++
+
+++
++++
7d
++++
+++
++++
++++
7e
++++
+++
++++
++++
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Chapter-6
Table 6.5: Antibacterial activity of compounds (8a-e)
% Zone of Inhibition
Sample
Gram +Ve
Gram –Ve
Bacillus
Staphylococcus
Klebsiella
E-Coli
subtilis
aureus
pneumoniae
8a
++
+
+
+
8b
++
+
++
++
8c
++
+
+
+
8d
+++
+++
++
++
8e
++
+++
++
++
Table 6.6: Antibacterial activity of compounds (9a-e)
% Zone of Inhibition
Sample
Gram +Ve
Gram –Ve
Bacillus
Staphylococcus
Klebsiella
E-Coli
subtilis
aureus
pneumoniae
9a
+
++
+
+
9b
+
++
++
++
9c
++
++
+
++
9d
++
+++
++
+++
9e
++
+++
++
+++
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Chapter-6
Table 6.7: Antibacterial activity of compounds (10a-e)
% Zone of Inhibition
Sample
Gram +Ve
Gram –Ve
Bacillus
Staphylococcus
Klebsiella
E-Coli
subtilis
aureus
pneumoniae
10a
+
++
++
++
10b
++
++
++
++
10c
++
+++
++++
+++
10d
+++
+++
++
+++
10e
+++
++++
+++
++++
Table 6.8: Antibacterial activity of compounds (11a-e)
% Zone of Inhibition
Sample
Gram +Ve
Gram –Ve
Bacillus
Staphylococcus
Klebsiella
E-Coli
subtilis
aureus
pneumoniae
11a
++
++
++
++
11b
++
++
++
++
11c
++
++
++
++
11d
++
+++
+++
+++
11e
+++
++++
+++
++++
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Chapter-6
6.3
Antifungal activity:
Following fungus were selected for fungicidal activity,
Rhizopus nigricans: is also known as bread mold. It found on spoiled food and in
soils.
Nigrospora sp: It is also carried and transferred from place to place through the soil,
the decaying plants and seeds
Aspergillus niger: It cause a disease called black mould on certain fruits and
vegetables such as grapes, apricots, onions, and peanuts. A.niger is less likely to cause
human disease which can cause pain, temporary hearing loss and in severe cases,
damage to the ear canal.
Method:
The antifungal activity of all the compounds (mentioned in earlier chapters) was
studied at 1000 ppm concentration in-vitro. Plant pathogenic organisms used were
Rhizopus Nigrican, Nigrospora Sp. and A. niger. Such activity of all the compounds
was measured on each of these plant pathogenic strains on a potato dextrose agar
(PDA) medium. This PDA medium was prepared from potato 200 gm., dextrose 20
gm., agar 20 gm. and water 1 liter. Five days old cultures were taken for study. The
compounds to be tested were suspended (1000 ppm) in a PDA medium and
autoclaved at 1200C for 15 min and at 15 atm. pressure. These media were poured
into sterile Petri plates and the organisms were inoculated after cooling the Petri
plates. The percentage inhibition for fungi was calculated after five days using the
formula given below.
Percentage of inhibition = 100 (1 – Y/X)
Where
X = Area of colony in control plate.
Y = Area of colony in test plate.
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The fungicidal activities displayed by various compounds are shown in
Tables: 6.9 to 6.16.
The Zone of inhibition data are presented in all tables as follows;
(+) = Zone of inhibition (≤ 59%), slightly active,
(++) = Zone of inhibition (60-64%), moderately active,
(+++) = Zone of inhibition (65-69%), highly active and,
(++++) = Zone of inhibition (≥ 70%), very high activity.
Table 6.9: Antifungal activity of Ketoconazole (standard)
Sample
Ketoconazole
Zone of inhibition at 1000 ppm (%)
Rhizopus nigrican
Nigrospora sp.
A. niger
++++
++++
++++
Table 6.10: Antifungal activity of compounds (5a-e)
Sample
Zone of inhibition at 1000 ppm (%)
Rhizopus nigrican
Nigrospora sp.
A. niger
5a
+
++
++
5b
+
++
+
5c
++
++
+
5d
+++
++++
++
5e
++++
+++
+++
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Chapter-6
Table 6.11: Antifungal activity of compounds (6a-e)
Sample
Zone of inhibition at 1000 ppm (%)
Rhizopus nigrican
Nigrospora sp.
A. niger
6a
++
+++
+++
6b
+
+++
++
6c
+++
++
++
6d
+++
++++
+++
6e
++++
++++
+++
Table 6.12: Antifungal activity of compounds (7a-e)
Sample
Zone of inhibition at 1000 ppm (%)
Rhizopus nigrican
Nigrospora sp.
A. niger
7a
+
++
++
7b
+
++
++
7c
++
++
+
7d
+++
+++
++
7e
+++
+++
+++
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Chapter-6
Table 6.13: Antifungal activity of compounds (8a-e)
Sample
Zone of inhibition at 1000 ppm (%)
Rhizopus nigrican
Nigrospora sp.
A. niger
8a
++
+
+
8b
++
+++
++
8c
++
++
+
8d
++++
++++
+++
8e
++++
++++
++++
Table 6.14: Antifungal activity of compounds (9a-e)
Sample
Zone of inhibition at 1000 ppm (%)
Rhizopus nigrican
Nigrospora sp.
A. niger
9a
+
++
++
9b
+
++
+
9c
++
++
+
9d
++++
+++
++++
9e
++++
++++
++++
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Chapter-6
Table 6.15: Antifungal activity of compounds (10a-e)
Sample
Zone of inhibition at 1000 ppm (%)
Rhizopus nigrican
Nigrospora sp.
A. niger
10a
+++
+++
+++
10b
+++
++
+++
10c
+++
+++
+++
10d
++++
++++
++++
10e
++++
++++
++++
Table 6.16: Antifungal activity of compounds (11a-e)
Sample
Zone of inhibition at 1000 ppm (%)
Rhizopus nigrican
Nigrospora sp.
A. niger
11a
++
+++
+++
11b
++
++
+++
11c
+++
+++
+++
11d
++++
++++
++++
11e
++++
++++
++++
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Chapter-6
6.4
Results and discussion:
The activity of all the newly compounds on Bactria and Fungi are shown in Tables:
6.1 to 6.8 and 6.9 to 6.16 respectively.
The compounds tested for antibacterial activity are listed in Tables-6.1 to 6.8 show
percentage (%) of zone of inhibition of bacterial growth of Gram-positive bacterial
strains B.subtilis and S.aureus and Gram-negative bacterial strains K. pneumoniae, S.
typhi and E.Coli.
Comparison of antibacterial activity of synthesized compounds with that of standard
antibacterial drug against all four bacterial strains.
Out of 3-((dialkylamino)methyl)-5-((2-((dimethylamino)methyl)-1H-benzimidazol-1yl) methyl)-1,3,4-oxadiazole-2(3H)-thione[5a-e] (Table 6.2) compounds 5e and 5d
show good antibacterial activity.
Among 4-(((1-((4-((dialkylamino)methyl)-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2yl)methyl)-1H-benzimidazole-2-yl)methyl)amino)-N-(pyrimidin-2-yl)benzene
sulfonamide[6a-e] (Table 6.3) compounds 6d and 6e show good antibacterial activity.
Compounds 7e and 7d show good antibacterial activity than the other compounds of
3-((dialkylamino)methyl)-5-((2-((ethyl(methyl)amino)methyl)-1H-benzimidazol-1yl)methyl)-1,3,4-oxadiazole-2(3H)-thione.[7a-e] ( Table 6.4)
In 3-((dialkylamino)methyl)-5-((2-((diethylamino)methyl)-1H-benzimidazol-1yl)methyl)-1,3,4-oxadiazole-2(3H)-thione[8a-e] (Table 6.5) compound 8d exhibit
good antibacterial activity.
In 3-((dialkylamino) methyl)-5-((2-((dipropylamino)methyl)-1H-benzimidazol-1yl)methyl)-1,3,4-oxadiazole-2(3H)-thione[9a-e] (Table 6.6), compounds 9d and 9e
show moderate antibacterial activity.
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Chapter-6
Among 3-((dialkylamino) methyl)-5-((2-((diphenylamino)methyl)-1H-benzimidazol1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione [10a-e] (Table 6.7) compound 10e show
good antibacterial activity.
In synthesized compounds, 3-((dialkylamino) methyl)-5-((2-((ethyl(phenyl)amino)
methyl)-1H-benzimidazol-1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione[11a-e]
(Table- 6.8) compound 11e show good antibacterial activity.
The compounds tested for fungicidal activity are listed in Tables-6.9 to 6.16
show Zone of inhibition at 1000 ppm (%) for Rhizopus Nigrican, Nigrospora Sp. and
A. Niger.
Comparison of antifungal activity of synthesized compounds done with Ketoconazole
as standard compound against all three fungal strains.
Among newly synthesized compounds 3-((dialkylamino)methyl)-5-((2-((dimethyl
amino)methyl)-1H-benzimidazol-1-yl) methyl)-1,3,4-oxadiazole-2(3H)-thione[5a-e]
(Table 6.10) compounds 5d and 5e show high antifungal activity.
Out of 4-(((1-((4-((dialkylamino)methyl)-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)
methyl)-1H-benzimidazole-2-yl)methyl)amino)-N-(pyrimidin-2-yl)benzene
sulfonamide[6a-e] (Table 6.11) compound 6d and 6e show good antifungal activity.
In 3-((dialkylamino)methyl)-5-((2-((ethyl(methyl)amino)methyl)-1H-benzimidazol-1yl) methyl)-1,3,4-oxadiazole-2(3H)-thione[7a-e] (Table 6.12) compounds 7d and 7e
show high antifungal activity.
In 3-((dialkylamino)methyl)-5-((2-((diethylamino)methyl)-1H-benzimidazol-1-yl)
methyl)-1,3,4-oxadiazole-2(3H)-thione[8a-e] (Table 6.13) compound 8d and 8e
exhibit good antifungal activity.
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Chapter-6
Out of 3-((dialkylamino) methyl)-5-((2-((dipropylamino)methyl)-1H-benzimidazol-1yl)methyl)-1,3,4-oxadiazole-2(3H)-thione[9a-e] (Table 6.14) compounds 9d and 9e
show good antifungal activity.
Among 3-((dialkylamino) methyl)-5-((2-((diphenylamino)methyl)-1H-benzimidazol1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione [10a-e] (Table 6.15) compounds 10d and
10e exhibit good antifungal activity.
Among 3-((dialkylamino) methyl)-5-((2-((ethyl(phenyl)amino)methyl)-1Hbenzimidazol-1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione[11a-e] (Table 6.16)
compounds 11d and 11e show good antifungal activity.
In conclusion all the compounds are toxic, more or less to selected bacteria as well as
fungi. More particularly the compounds d and e in all series are more toxic, this might
be responsible due to presence of phenyl ring.
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Chapter-6
References:
[1]
B.P.Patel,P.J.Shah and H.S.Patel, J.Saudi chemical Soc.,17,307 (2013).
[2]
A.R.Shah, H.R.Dabhi and A.K.Rana.,Der Pharma Chemica, 7(3),158(2015)
[3]
J.D.Bhatt, K.S.Nimavat and K.B.Vyas., J.Chem.Pharm.Res., 5(10),327(2013).
[4]
M.Sharda and G.D.Acharya., Der Pharma Chemica, 7(8), 25(2015)
[5]
C. Robert, “Medical Microbiology”, ELBS, Livingston, 11th edition,.815, 901
(1970).
[6]
G. D. Sujatha, Ind. J. Expt. Biol., 13, 286 (1975).
[7]
R.J.Patel and K.S.Trivedi, Experimental Microbiology,Aditya Publication,7th
Edition (2014)
[8]
S.A.Walksman, “Microbial Antagonism and Antibiotic Substances”,
Commonwealth Fund, N.Y., 2nd edition, 72 (1947)
[9]
R.Cruickshank, J.P.Dugid, D.P.Marmion and R.H.A.Swain,"Medical
Microbiology”, Churchil-Livingstone, Edinburgh, London, Vol. 2, 12th edition
(1975).
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