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Chapter X
ANTmACTERIAL AND ANTIFUNGAL ACTIVITIES OF SOME
LANTHANIDE(III) CHLORIDE AND NITRATE COMPLEXES OF
2-(N-INDOLE-2-0NE)AMINO-3-CARBOXYETHYL-4, 5, 6, 7
TETRAHYDROBENZO[blTHIOPHENE
From the inception of civilization, human being struggles for
existence against the affliction of disease, decay and death. It is the eternal
want of human to remain healthy and cure from his surroundings. But in
the ancient era, millions of people died from various infectious diseases
like plague, cholera, diarrhea, tuberculosis etc, in epidemic form, which has
instigated the man to endeavor for remedy for their sufferings. In fact in the
way of long struggle for relief from many infectious diseases as well as
illness, physical discomforts, injuries, pain and wounds, man had achieve
the mastery over the diseases specially infectious diseases when Alexander
Fleming accidentally discovered penicillin from a microorganism.
Although antibiotics are life saving drugs but nowadays, because of
careless and promiscuous use of antibiotics, various pathogenic microbes
are gaining resistance. To overcome this problem and to treat many serious
infectious diseases like AIDS, cancer, viral fever, continuous search of new
228
more potent antibiotic is indispensable. A large number of organic
compounds have been developed as antimicrobial agents. Development of
resistance among different species of pathogenic bacteria has given new
dimension to this area.
!satin and its derivatives are unique among the organic compounds
mainly because of their biological significance. Apart from the synthetic
flexibility with which they can be transformed into other derivatives, they
form an important class of compounds which possess activity against
. 325 326 1 327 fu 1 328-331 . 1332 dtuberculosis, ' eprosy, nga , vua an bacterial333•334
, . h
· 335 · · 336 d
t' 1 t 337-339 mfectlons, r eumatlsm, trypanosom1as1s an as an 1convu san s.
Similarly thiophene and its analogs represent an important class of
compounds with diverse biological activities.340 Substituted thiophenes
h . I d. . h 341ave potentia ra 10-protectlve c aracter, 1 · 3
42ana ges1c, antitussive,
antiinflammatory, bactericidal, fungacidal etc activities. Schiff bases play
an outstanding role in many biological processes.343 Their antimicrobial
activities have been reported to increase on coordination with metal ions.
These two biologically important compounds have been incorporated
through an azomethine functionality and the resulting ligand and thus metal
chelates have been examined as antibacterial and antifungal agents. Metal
chelates of sulphur containing Schiff bases have received major attention
229
of biochemists because of their versatile use as antimicrobial agents.344
Compounds containing C=S and c=o groups possess fairly good
fungitoxicity.345,346 There are recent reports on the antimicrobial activities
of lanthanide(III) complexes.347,348 All these observations prompted us to
synthesise some lanthanide(III) Schiff base complexes of 2-(N-indole
2-one)amino-3-carboxyethyl-4,5,6,7-tetrahydrobenzo[b]thiophene and
enticed to the evaluation of the antibacterial and antifungal activities. So
this chapter is solely concerned with antibacterial and antifungal activities
of lanthanum(III), cerium(III), praseodymium(III), neodymium(III),
samarium(III), europium(III), gadolinium(III), dysprosium(III),
ytterbium(lII) and lutetium(III) nitrate and chloride complexes of
2-(N-indole-2-one)arnino-3-carboxyethyl-4,5,6,7-tetrahydrobenzo[b]
thiophene synthesised during this investigation.
EXPERIMENTAL
Antibacterial and antifungal activities of lanthanum(III) chloride and
nitrate complexes of 2-(N-indole-2-one)amino-3-carboxyethyl-4,5,6,7
tetrahydrobenzo[b]thiophene were studied against the four bacteria
Escherchia coli, Vibrio cholerae, Salmonella typi and Bacillus megaterium
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and the four fungi Penicillium notatum, Aspergillus niger, Candida
lb ' d S h .. b d'ff' h d 193 194349a leans an ace aromyces cerevlszae y agar 1 uSlOn met o. ' ,
Escherchia coli
Escherchia coli is a type of fecal coli form, Gram negative and non-
sporing bacteria commonly found in the intestines of animals and human.
The virulent strains of Escherchia coli act as specific pathogens of the gut
(enteritis) and of extra-intestinal sites (urinary tract infection, wound
infection). Most strains are mobile by peritrichalate flagella. Regarding
biochemical reaction they break down most of the sugars. Infection often
causes severe bloody diarrhea and abdominal cramps. They are usually
sensitive to many antibiotics such as ampicillin, tetracyclin,
chloramphenicol, gentamycin etc. They are aerobic and facultative
anerobes and grow in ordinary culture media (nutrient broth, nutrient agar)
at 37°C in 8 to 24 h.
Vibrio cholerae
Vibrios are gram negative, highly motile curved rods with a single
polar flangellum and appear to 'vibrate'. Some are pathogenic. Of the
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vibrios that are chemically significant to human, Vibrio cholerae, the
agent of cholera, is the most important. Most vibrios have relatively simple
growth factor requirements and will grow in synthetic media with glucose
as a sole source of carbon and energy. However, since vibrios are typically
marine organisms, most species require 2 - 3% NaCl or a sea water base for
optimal growth. In liquid media vibrios are motile by polar flagella. These
organisms are sensitive to acid pH but tolerate alkaline pH (9 - 9.6) very
well.
Salmonella typi
Salmonella typi is a Gram negative facultative rod shaped bacterium
in the same protea-bacterial family, trivially known as 'enteric' bacteria. It
causes typhoid fever and it can be transmitted by the fecal-oral route. It is
excreled by human in feces and may be transmitted by contaminated water,
food or by person - to - person contact. Growth temperature is minimum at
7 °C, growth greatly reduced at < 15 °C, maximum at 45 °C and optimum
at 35 - 37 °C. Optimum pH is 7 - 7.5.
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Bacillus megaterium
Bacillus megaterium is a gram positive spore producing bacteria. It
is a rod shaped Eubacteria and is found in the soil. Colonies form in chains
due to sticky polysaccharides on the cell wall. It is very important in the
biotech industry due to its size and its' enzyme and cloning ablities.
Penicillium notatum
Penicillium notatum, also known as Penicillium chryogenum, is the
blue - green mold on bread, fruits and nuts. It grows at its peak during the
spring and the winter, but the mold has no seasonal variations. It is a well
known group of brushed - shaped microorganism and is heterotropic.
Aspergillus niger
Aspergillus niger is a fungus and one of the most common species of
the genus Aspergillus. It causes black mold on certain types of fruit and
vegetables and is a common contaminent of food. It is less likely to cause
disease than some other Aspergillus species, but if large amounts of spores
are inhaled, a serious lung disease aspergillosis can occur. It is one of the
most common causes of otomycosis (fungal ear infections), which can
233
cause pain, temporary hearing loss and in severe cases damage to the ear
canal and tympanic membrane.
Candida albicans
Candida albicans is a diploid sexual fungus and a casual agent of
opportunistic oral and vaginal infections in humans. Systemic fungal
infections have emerged as important causes of morbidity and mortality in
immunocompromised patients (eg. AIDS, cancer, chemotherapy, organ or
bone marrow transplantation). It is among the many organisms that live in
the human mouth and gastrointestinal tract. Under normal circumstances, it
lives in 80 % of the human population with no harmful effects, although
overgrowth results in candidiasis.
Saccharomyces cerevisiae
Saccharomyces cerevisiae commonly known as bakers yeast' is a
single celled ascomycete. Widely used as a simple eukaryotic model,
particularly in recombinant DNA and cell cycle studies as well as in mating
type and hetrokaryon compatibility studies. It can reproduce both asexually
and sexually; and can be cultured in either the haploid or the diploid state.
234
Microorganisms are ubiquitous. They are found in soil, air, water,
food, sewage and on body surfaces. The microbiologist separates these
mixed populations into individual species for study. A culture containing a
single unadulterated species of cells is called a pure culture. The survival
· and continued growth of microorganisms depend on an adequate supply of
nutrients and a favourable growth environment. For the former, most
microbes must use soluble low - molecular weight substances that are
frequently derived from the enzymatic degradation of complex nutrients is
a culture medium. Basically, all culture media are liquid, semisolid or solid.
A liquid medium lacking a solidifying agent is called a broth medium. A
broth medium supplemented with a solidifying agent called agar results in a
solid or semisolid qiedium. Agar serves as an excellent solidifying agent
because it liquefies at 100 °C and solidifies at 40 °C. Because of these
properties, organisms, especially, pathogens can be cultivated at
temperatures of 37 .5 °C or slightly higher without fear of the medium
liquefying. A completely solid medium requires an agar concentration of
about 1.5 % to 1.8 %. A concentration of less than 1 % agar results in a
semisolid medium. A solid medium has the advantage that it presents a
hardened surface on which microorganisms can be grown using specialized
techniques for the isolation of discrete colonies. Each colony is a cluster of
235
cells that originates from the multiplication of a single cell and represents
the growth of a single species of microorganism. Such a defined and well -
isolated colony is a pure culture. Also, while in the liquefied state, solid
media can be placed in test tubes, which are then allowed to cool or harden
in a slanted position, producing agar slants. These are useful for
maintaining pure cultures.
Nutrient broth
It was prepared by dissolving peptone (1 g), meat extract (0.5 g) and
sodium chloride (0.5 g) in distilled water (100 mL). The pH of the solution
was adjusted to 7 .2 -7.4.
Nutrient agar
This medium is used for maintaining pure bacterial culture and to
lawn the bacteria for detecting antibacterial activity. It was prepared by
dissolving peptone (0.5 g), meat extract (0.2 g), sodium chloride (0.5 g)
and agar (2.5 g) in distilled water (100 mL). The pH of the medium was
maintained at 7 .2 - 7.4.
236
Subculture
One day prior to the test, the bacteria were inoculated in nutrient
broth (inoculation medium) and kept in an incubator at 37°C for 24 h.
Seed layer medium
The bacterial subculture was taken in the seed layer medium for
lawning. It consist of peptone (l g), yeast extract (0.8 g), glucose (0.2 g),
sodium chloride (0.6 g) and agar (0.2 g) in distilled water (100 mL) and
sterilized.
Compound solution
A stock solution of the ligand and the complexes were prepared by
dissolving 0.5 mg of the compounds in 1 mL of DMSO so that their
concentration is 500 ~ g / mL.
Zone of inhibition
When a filter paper disc, impregnated with the compound solution is
placed on agar the compound will diffuse from the disc to the agar. This
diffusion will place the compound in the agar only around the disc. The
237
solubility of the compound and its molecular size will determine the size of
the area of chemical infiltration around the disc. If an organism is placed on
the agar it will not grow in the area around the disc if it is susceptible to the
chemical. This area of no growth around the disc is known as a zone of
inhibition'.
Antibacterial activity
The antibacterial activity of ISAT and its chloro and
nitrato complexes of lanthanum(III), cerium(III), praseodymium(III),
neodymium(III), samarium(III), europium(III), gadolinium(III),
dysprosium(III), ytterbium(III) and lutetium(III) were evaluated by the agar
diffusion method. The hot nutrient agar solution (20 rnL) was poured into
sterilized petri dishes and allowed to attain room temperature. The seed
layer medium was melted and cooled to about 45 °C with gentle shaking.
The previously grown subculture was added to the seed layer medium
aseptically and mixed well. It was immediately lawned into petri dishes and
allowed to attain room temperature. Filter paper discs (6 mm dia.) were
soaked in solution of the test compounds in DMSO and placed after drying
off the solvent on petri dishes containing lawn culture of different bacteria.
The plates were incubated to an optimum temperature of 37 °C for 48 h. At
238
the end of incubation period, the zones of inhibition around the discs were
measured. Gentamycin was used as reference standard.
Antifungal activity
Antifungal activity of the ligand and the complexes was tested by
the agar diffusion method using Flucanazole as the standard. Instead of
agar petri dishes in antibacterial study, agar slants prepared in sterilized test
tubes were used. The test tubes were closed with cotton plugs to maintain
sterile ervironment. Incorporation of the test compounds in SDA
(sabouraud dextrose agar) inoculation of the fungal culture, the presence,
absence or degree of growth were determined.
RESULTS AND DISCUSSION
Antimicrobial activities of the ligand, ISAT and its chloro and
nitrato lanthanide(III) complexes against the bacteria and fungi mentioned
above were studied and the results have been presented in Table X. 1 to
Table X. 4 and Fig X. 1 to Fig X. 3. It is interesting to observe that
the complexes are more potent bactericide and fungicide than the ligand.
This is in consistent with the reports that antimicrobial properties of organic
239
compounds are considerably enhanced by complexation with lanthanide
(III) ions. 350-352
A possible mode of toxicity can be speculated in the light of.
chelation theory. 353 Chelation reduces the polarity of the metal ions
considerably, mainly because of the partial sharing of its positive charge
with donor groups and possible 1t-electron delocalization on the whole
chelate ring. The lipids and polysaccharides are some important
constituents of cell wall and membranes, which are preferred for metal ion
interaction. In addition to this, cell wall also contains many
aminophosphates, carbonyl and cysteinyl ligands, which maintain the
integrity of the membrane by acting as a diffusion barrier and also provides
suitable sites for binding. Chelation can reduce not only the polarity of the
metal ion, but it increases the lipophilic character of the chelate and the
interaction between metal ion and the lipid is favoured. This may lead to
the breakdown of the permeability barrier of the cell resulting in
interference with the normal cell processes.
Chelation is not the only criterion for antibacterial activity.354 Some
important factors which determine the activity are, nature of the metal ion,
nature of the ligand, coordinating sites, geometry of the complex,
concentration, hydrophilicity, lipophilicity and presence of co-ligand. Steric
240
Table X. 1 Antibacterial activity of ISAT and [Ln(ISAT)(N03)3]
Inhibition zone (mm)
Compound Escherchia Vibrio Salmonella Bacillus
coli. cholerae typhi megaterium
!SAT 6 7 8 7
[La(ISA T)(N03)3] 8 10 12 11
[Ce(ISA T)(N03)3] 9 11 14 12
[Pr(ISA T)(N03)3] 10 12 13 14
[Nd(ISAT)(N03)3) 11 11 15 13
[Sm(ISAT)(N03)3) 10 12 17 15
[Eu(ISAT)(N03)3) 12 14 18 14
[Gd(ISAT)(N03)3) 11 15 16 13
[Dy(ISA T)(N03)3) 10 14 15 12
[Yb(ISAT)(N03)3) 12 16 14 15
[Lu(ISA T)(N03)3) 11 15 13 14
241
Table X. 2 Antibacterial activity of ISAT and [Ln(ISAT)Cb]
Inhibition zone (mm)
Compound Escherchia Vibrio Salmonella Bacillus
coli. cholerae typhi megaterium
ISAT 6 7 8 7
[La(ISAT)Ch] 10 12 14 13
[Ce(ISAT)Ch] 11 13 15 14
[Pr(ISAT)Ch] 12 14 14 13
[Nd(ISAT)Ch] 12 15 16 14
[Sm(ISA T)Cl3] 13 14 15 13
[Eu(ISA T)Ch] 14 16 18 15
[Gd(ISAT)Ch] 12 15 16 14
[Dy(ISAT)Ch] 11 16 17 15
[Yb(ISA T)Ch] 13 16 17 14
[Lu(ISAT)Ch] 12 15 16 13
242
243
Table X. 4 Antifungal activity of ISAT and [Ln(ISAT)ChJ
% Activity
CompoundP. notatum A. niger C. albicans S. cerevisiae
ISAT +++ +++ +++ +++
[La(ISAT)ChJ + + + +
[Ce(ISAT)CI3J + + + +
[Pr(ISAT)CI3J +
[Nd(ISAT)CI3J + +
[Sm(ISAT)ChJ + + + +
[Eu(ISAT)CI3J + + + +
[Gd(ISAT)ChJ +
[Dy(ISAT) ChJ +
[Yb(ISAT)CI3J + +
[Lu(I AT)ChJ + +
25% inhibition: +++ 50% inhibition: ++ 75% inhibition: + 100% inhibition:
244
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246
Fig X.l
Antibacterial activity
247
Fig X.2
Full growth of fungi
248
Fig X.3
Antifungal activity
249
and pharmacokinetic factors also play important roles in deciding the
potency of an antimicrobial agent. The interaction of metal ions with
cellular compounds is due to the fact that all these structures contain a
variety of functional groups that can act as metal-binding sites. The
difficulty is how to obtain these interactions in cells and organisms where
non-polar membranes exist to hinder the movement of charged metal ions
in to the cell, where a myriad of metal-binding sites exist to complete for
the metal ion. In order to achieve this, the complex should possess
sufficient lipid solubility to permit transport of the metal across the
membranes and it must have high thermodynamic stability to reach the site
without being dissociated. Finally, it can act as metal complex with cells
to produce a selective effect in order to obtain therapeutic value.355
Role of co-ligands on antimicrobial activity was also examined. On
comparing the activities of lanthanide nitrate and chloride complexes it has
been observed that the chloride complexes exhibited more inhibition. The
bonding capacity of nitrate ion towards lanthanide ion is greater than that of
the chloride ion. As a result of this the extent of metal ion available in
lessened for the display of antimicrobial activity. Besides this mode of
action, the compounds may indulge in the formation of hydrogen bonded
interaction throu ah the coordinated anion, azomethine group etc. with theb
250
active centres of the cell constituents resulting in the interference with the
normal cell processes. Hence it can be concluded that antimicrobial activity
possessed by metal complexes is an intricate blend of several contributions.