Evaluation Of Antibacterial , Antifungal And

Anti-inflammatory Activity Of Novel Pyrazole

Linked Thiazolidinedione Derivatives

Presented by:
SUMAYYA ZAFFAR, B.Pharm.
170116887002

Under guidance of :
Dr B.Veeresh, M.Pharm,Ph.d.
Professor
Department of Pharmacology
G.Pulla Reddy College of pharmacy
1
 Contents
• Introduction

• Review of Literature

• Aim and Objectives

• Materials & Methods

• Results and Discussion

• Summary and Conclusion

• References

2
 Introduction
Bacteria are unicellular free living organisms having both DNA
and RNA. They are capable of performing all essential processes
of life, e.g., growth, reproduction, and metabolism.

3
Classification Of Bacteria

4
Fig. 2: Diseases caused by
various bacteria in
humans

5
Mechanism of Action of Antibacterial agents

6
FUNGI:
 The fungi are eukaryotic organisms and they differ from the
bacteria, which are prokaryotic organisms, in many ways. The fungi
possess rigid cell walls, which possess two characteristic cell
structures: chitin and ergosterol.

7
 Table 1: Infectious Diseases Caused by Fungi

Diseases Etiolgical agents Main tissue affected

Contagious, Superficial agent
Dermatophytoses Epidermophyton, skin, hair, nail
Microsporum,
Non contagious, Systemic diseases
Aspergillosis Aspergillus spp. external ear, lungs, eye, brain
Blastomycosis Biastomyces dermatitis lungs, skin, bone, testes
Candidiasis Candida respiratory, gastrointestinal and urogentital
tracks, skin
Choromomycosis Cladosporum Skin
fonsecaea and
Phidlophora spp.
Coccidioidomycosis Coccidioides immitis lungs, skin, joints, meninges
Cryptoccosis Cryptococcus neoformans lungs, meninges
Histoplasmosis Hipstoplasma capsulatum lungs, spleen, liver, adrenals, lymph
nodes
Mucormycosis Absidia, Mucor, nasal mucosa, lungs, blood vessels, brain
Rhizopus spp
8
Antifungal agents and primary sites of activity

9
INFLAMMATION:
•A localized physical condition in which part of the body
becomes reddened, swollen, and often painful, especially as a
reaction to injury or infection.

• It is part of the complex biological response of body tissues

to harmful stimuli, such as pathogens, damaged cells, or
irritants, and is a protective response involving immune cells,
blood vessels, and molecular mediators.

10 10
 Phases of inflammation
Acute : vasodilation & increased capillary permeability.

Delayed: infiltration of leukocytes and phagocyte cells.

Chronic proliferative: tissue degeneration and fibrosis.

Mediators of inflammation

12
 THIAZOLIDINEDIONES (TZD’S):

Thiazolidinediones are sulfur containing pentacyclic compounds that are

surplusly found in nature in various forms. Thiazolidinedione nucleus is present
in numerous bioactive compounds, e.g., anti-malarial, antimicrobial, anti-
mycobacterium, anticonvulsant, antiviral etc.

Owing to the rapid development of new molecules containing this nucleus,

many research reports have been generated in a brief span of time.

Resulting in strong requirement to collect recent information in order to

understand the current status of the thiazolidinedione nucleus in medicinal
chemistry research.

Thiazolidinedione moeity

13
Most of thiazolidinediones exhibit good bactericidal activity against various Gram-
positive and Gram-negative bacteria. The bactericidal activity of thiazolidinediones
derivatives depends on the substitution on the heterocyclic TZD ring rather than the
aromatic moiety.

TZDs, such as glitazones (ex: rosiglitazone and pioglitazone), are involved in

lowering of plasma glucose levels by acting as ligands for the gamma peroxisome
proliferator-activated receptors (PPARs).

Pyrazoles were reported to possess a wide range of biological activities in literature

such as anti-microbial, anti-fungal, anti-tubercular, anti-inflammatory etc.The high
profile NSAID celecoxib, contains the pyrazole nucleus. This cyclooxygenase
(COX)-2 inhibitor is considered to be a potent anti-inflammatory and analgesic agent.

Owing to the biological significance of these two classes of compounds many

researchers have, synthesized and extensively studied a combined molecular
framework that involves these two different bioactive scaffold.

14
 Review of Literature
AUTHOR DERIVATIVES ORGANISMS ACTIVITY
Nisheeth.C Thiazolidine-2,4-dione S. aureus, S. epidermidis, Antimicrobial
desai (2014) pyrazole moiety E. coli, K. pneumoniae,
P. aeruginosa,
Salmonella typhi,
Amal.M Pyrazolyl-2,4- Staphylococcus.aureus, Antimicrobial,
yousef., thiazolidinediones Escherichia coli, Candida Anti-
(2010) albicans inflammatory,
Anticancer
Ming–xia Rhodanine-based-5- Methicillin resistant and Antibacterial
song aryloxy pyrazole quinolone resistant
(2012) staphylococcus aureus

Lingaiah Benzosuberone,bearing Anti cancer.

nagarapu 2,4-thiazolidenone moeity
(2013)

15
Aim & Objectives
Aim & Objectives

Aim:
The aim of the study was to evaluate Antibacterial , Antifungal and
Anti-inflammatory activity of novel Pyrazole linked thiazolidinedione derivatives.

Objectives:

 Screening and selection of microorganisms.

 Evaluation of antibacterial activity- agar well diffusion and suspension method
 Evaluation of antifungal activity- agar well diffusion and suspension method
 Evaluate anti-inflammatory activity- HRBC membrane stabilization method.

16
 Materials & MethodS
* NOVEL PYRAZOLE LINKED THIAZOLIDINEDIONE DERIVATIVES :
S.no Compound code Structure

1. A

2. B

3. C

17
4. D

5. E

6. F

7. G

18
 Materials for Antibacterial Activity

• Muller Hinton Glassware

Broth
• Ciprofloxacin Petri plates, test tubes, cotton
Himedia Discs (std) swabs, inoculum

• Agar Agar
• DMSO 2.5%
Finar (GPRCP) Equipments
Autoclave, incubator, hot air
oven, laminar air flow
• Pyarazole-linked
Dr. Subhashini TZD derivatives
(Osmania
university)

19
 Materials for Antifungal Activity

• Potato dextrose infusion Glassware

medium at pH (5.6±0.2) Petri plates, test tubes, cotton
Himedia
swabs, inoculum

• Agar Agar
Finar • DMSO (control )

Equipments
Autoclave, incubator, hot
• Itraconazole Tablets
Apollo air oven, laminar air flow
Pharmacy

• Pyrazole-Linked TZD
Derivatives
Dr.Subhashini
(Osmania
University)
20
 TEST CULTURES
Following common standard strains were procured from National Chemical
Laboratory, Pune :
Bacterial strains:
Escherichia coli [Gram negative] (NCIM-5011)
Pseudomonas aeruginosa [Gram negative] (NCIM-5029)
Staphylococcus aureus [Gram positive] (NCIM-2079)
Bacillus subtilis [Gram positive] (NCIM-2063)
Fungal strains :
Aspergillus.niger
Pencillium.notatum

Preparation
Preparation Preparation
of Nutrient Preparation
of Test of Drug
Broth for of medium
Inoculums Solution
inoculation

21
 Materials for anti-inflammatory activity

Test drug: Thiazolidinedione compounds (Osmania University )

Standard drug: Diclofenac Sodium (Apollo Pharmacy)

Iso-saline solution: (0.9% Nacl)

Alseviour solution: (Dextrose 2.5%, sodium citrate 0.8%, citric acid

0.5%, Nacl 0.42%)

Hyposaline solution: (0.25%Nacl)

22
 Agar Well Diffusion Method for Estimation of
Zone of Inhibition
The solidified agar plates were inoculated by dipping a sterile swab into inoculums.
Excess inoculum was removed by pressing and rotating the swab firmly against the
side of the tube, above the level of the liquid.

The swab was streaked all over the surface of the medium three times, rotating the
plate through an angle of 60o after each application. Finally the swab was passed
round the edge of the agar surface. The inoculation was dried for a few minutes, at
room temperature, with the lid closed.

Ditch the bore in plate. 50 μl of test and control solutions was added in respective
bore.

Standard ciprofloxacin disc were placed on agar plate using sterile forceps.
23
The plates were kept undisturbed for atleast 30 mins at room temperature to allow
proper diffusion of the test and standard solution into the agar medium.

The above procedure was conducted aseptically in laminar air flow work station.

The plates were then placed in an incubator at 37oC for 24 hrs.

After 24 hrs incubation for bacteria, the diameter of zone (including the diameter
disc) was measured and recorded in mm. The measurements were taken with a
ruler, from the bottom of the plate, without opening the lid.

24
 Suspension Method for Estimation of
concentration of Inhibition

In this method a suspension of 150µL medium and organism was added in wells of
a microtitre plate

Test Drugs From A to G were then added at 15µL and standard drug , 5µL of
ciprofloxacin and 2.5% DMSO was used as control

Without disturbing the plate it was covered with the silver foil and incubated at
37oC for 24 hours and the same was followed for antifungal activity-
21o C for 48hrs

25
 The concentration of inhibition was then observed in comparison with the
standard drug and based upon the activity (indicative of turbidity), test drugs
were sorted out.

The above compounds were then carried out for finding out the minimum
inhibitory concentration (MIC)

26
Determination of Minimum Inhibitory Concentration
(MIC) for Antibacterial and Antifungal Activity.

Two fold dilution of the test compounds and of standards i.e. ciprofloxacin for
antibacterial activity and Itraconazole for antifungal activity were prepared in
Mueller Hinton broth and Potato Dextrose infusion respectively.

The test compounds (2mg) were dissolved in DMSO (1ml) to obtain required
concentration of 1, 3, 6, 12 & 25 µg/ml.

The bacterial inoculate were prepared by suspending 24 hr old bacterial colonies

from MH broth and fungal inoculate were prepared by suspending 48 hr old
fungal colonies from potato dextrose infusion .

27
 Suspensions were then diluted in 0.85% saline to give 107 CFU/ml.

Test tubes were spot-inoculated with 1µl of each prepared bacterial and fungal
suspension (104CFU/spot), which was further incubated at 37°C for 24h and 21°C
for 48hrs respectively.

At the end of incubation period, MIC was determined by turbidimetric method ,

which is the lowest concentration of each test compound that resulted in no visible
growth in the test tubes.

In order to ensure that the solvent had no influence on microbial growth, the
control test was performed with test medium supplemented with DMSO at the
same dilutions as used in the experiment.

28
Micro Titre plates with 96 wells:

29
Anti-inflammatory activity:
Standard and Test drugs were divided into following concentrations:

Concentrations

1μg/ml 3μg/ml 100μg/ml 300μg/ml

30
PROCEDURE:

Blood was collected from healthy human who had not taken any NSAIDS
for 2 weeks prior to the experiment

The blood was mixed with equal volume of Alsevier solution (Dextrose
2.5%, sodium citrate 0.8%, citric acid 0.05%, Nacl 0.42%) and distill water
100ml and centrifuged at 3000 rpm ,packed cells were washed with iso
saline

To 1ml HRBC suspension equal volume of test drug in four different conc.
1,3,100,300 µg/ml was added

Diclofenac sodium was used as a standard and iso-saline was taken as

control

31
 The assay mixtures were incubated at 37oC for 30 minutes and centrifuged

The haemoglobin content in the supernatant solution was estimated using UV

analysis at 560nm

The percentage protection/inhibition was calculated

32
The percentage of HRBC membrane stabilization or protection was
calculated :

Formula = Abs of test solution - Abs of control

Abs test solution

33
 Mechanism Of Anti-inflammatory activity:

Human red blood cell stabilization method.

PRINCIPLE : Lysosomal enzyme release Anti-inflammatory dugs
(proteases, nucleases etc) -

Acute/chronic inflammation

Anti-inflammatory dugs
Destabilization or lysis of HRBC -
membrane (Hypotonicity,heat etc)

Anti-inflammatory activity
34
 Results and Discussion
Antibacterial activity :
1. Agar well diffusion method: The zone of inhibition was not observed by this method
2. Suspension method : The Minimum inhibitory concentration for the test
compounds was obtained as follows:

Minimum inhibitory concentration (MIC) (μg/ml)

Compound code Gram Negative Gram Positive
E. coli P. aeruginosa S. aureus B. subtilis

A 6 6 12 1
B 6 12 12 12
C 3 12 12 12
D 6 6 12 6
E 1 6 12 6
F 12 6 12 12
G 6 12 12 6
Standard
5 5 5 5
(Ciprofloxacin)
35
 Antifungal activity:
1. Agar well diffusion method: The zone of inhibition was not observed by this method
2. Suspension method : The Minimum inhibitory concentration for the test
compounds was obtained as follows:

MIC (µg/ml) MIC (µg/ml)

Compound code
Asperigillus.n
A 12
B 12
C 6
D 6
E 6
F 12
G 12
Standard
(Itraconazole) 10
Compound code
Penicillium.n
A 12
B 6
C 12
D 12
E 3
F 12
G 12
Standard
(Itraconazole) 10
36
Absorbances of test drugs at different concentrations:

S.no Compound code Concentrations (µg/ml)

1 3 100 300

1 A 0.24 0.18 0.10 0.12

2 B 0.25 0.23 0.20 0.16

3 C 0.24 0.10 0.07 0.05

4 D 0.25 0.23 010 0.11

5 E 0.20 0.15 0.07 0.05

6 F 0.18 0.11 0.09 0.04

7 G 0.15 0.12 0.17 0.06

8 DICLOFENAC SODIUM(Std) 0.1 0.09 0.07 0.02

9 CONTROL 0.342

37
 Anti-inflammatory activity:

S.no Sample name % of Inhibition

Concentration (μg/ml)

1 3 100 300

1 A 50 48 65 73

2 B 31 32 41 69

3 C 58 72 85 88

4 D 31 28 65 72

5 E 45 62 64 65

6 F 47 57 55 68

7 G 56 64 70 72

8 Diclofenac sodium (STD) 73 74 79 94

38
 100

90

80
Concentrations (ug/ml)
70
% INHIBITION

60 1

50 3

40 100

300
30

20

10

0
Diclofenac A B C D E F G

 When compared to standard (Diclofenac) compound ‘C’ has shown maximum

percentage of inhibition but not as potent as Diclofenac .

Compounds A,B,D,E,F and G have shown less than 80% of inhibition which indicate
that they have mild to moderate anti-inflammatory activity .
39
 DISCUSSION
Antibacterial and Antifungal Activity:

The study involved antibacterial and antifungal testing against against a panel of
selected gram positive (B.subtilis and Staphylococcus aureus) and gram
negative bacteria (E.coli &Pseudomonas aerogenosa) as well as the fungal
strains (Aspergillus niger and Pencillium notatum)in comparison with the
reference drugs ciprofloxacin and itraconazole respectively .

 The absence/low turbidity at the bottom of the well containing culture medium
was indicative of inhibition of bacterial and fungal growth.

The compounds C&E exhibited good antibacterial activity with an MIC of 3 and
1 µg/ml against E.coli comparable to that of standard drug.The compound A
showed more potent inhibition with MIC 1µg/ml against gram positive
bacteria,B.subtilis.

The compounds B,C,D&E, exhibited good antifungal activity against the fungal
starins.

40
*Derivatives having substitution at- C6H5 group is thought to be responsible for this
antibacterial and antifungal activity of the the tested drugs

*Khan et al. (2016) designed some novel diphenyl tetrazole thiazolidinedione

derivatives and evaluated for their antimicrobial activity against bacterial strain
(Escherichia coli, Bacillus subtilis).The results indicated that most of the
synthesized derivatives showed potent invitro antimicrobial activity.

*Sharma et al. (2012) synthesized a series of novel N-(-5-arylidene-2-(4-

chlorophenyl)-4-oxothiazolidin-3-yl) derivatives by knoevenagel condensation and
assayed for antibacterial activity against Escherichia coli, Staphylococcus
aureus, Bacillus subtilis & anti fungal activity against Aspergillus
niger, Saccharomyces cervesia using turbidimetric method.The synthesized
compounds resulted in wide spectrum antimicrobial activity against all the tested
bacteria and fungi using ciprofloxacin and clotrimazole as a standard drug
respectively.

*Furthermore the data obtained indicates that among the derivatives the inhibitory
affect is dependent on substitution at thiazolidinedione ring. The introduction of
halogen atoms on the phenyl ring improved both antifungal and antibacterial
activity. 41
 Anti-inflammatory activity: :
The study investigated the anti-inflammatory activity of pyrazole linked TZD
derivatives using HRBC stabilization method.

This method was used because the erythrocyte membrane resembles the
lysosomal membrane and as such, the effect of drugs on the stabilization of
erythrocyte membrane could be extrapolated to the stabilization of lysosomal
membrane. Therefore as a membrane stabilizer,it interferes with the release and/or
action of mediators like histamine, serotonin,prostaglandins, and leukotrienes.(J.V
Sirisha.et.al, 2013)

The test drugs at concentrations 1, 3, 100, 300µg/ml, protected the human

erythrocyte membmranes against lysis induced by hypotonic solution.All the
compounds have shown mild to moderate anitiinflammatory activity .

The compound ‘C’ has shown highest percentage of inhibition but not as potent
as the standard drug Diclofenac sodium.

42
The substitution of chloro atom on – C6H5 ring attached to pyrazole moiety is
thought to be responsible for its anti-inflammatory activity.

It is well established that both Non-steroidal anti-inflammatory and steroidal

ant-inflammatory drugs protect erythrocyte membranes from hypotonicity
induced hemolysis.

Several studies have reported invitro anti-inflammatory activity of various

compounds using HRBC membrane stabilization method.

Shazia .T et al. (2017) have reported invitro anti-inflammatory activity of

Euphorbia wallichi against four different concentrations of standard drug
Diclofenac sodium.

Maria .d et al. (2016) have reported invitro anti-inflammatory activity of

Thiazolidine-4-one derivatives of ferulic acid using HRBC method compared
with different concentrations of Diclofenac sodium as standard drug.

43
The investigation suggested good ability of pyrazole linked TZD derivatives to
resist the cell lysis in small concentrations as compared to the standard drug, though
not greater than Diclofenac.

This invitro method was more time saving, flexible,and convenient in many ways.

As regards the relationships between structure and the detected antibacterial
antifungal and anti-inflammatory activity, the chlorophenyl, flourophenyl and
diphenyl derivatives showed significant results greater than that of the parent
compound (Thiazolidine,2-4-dione) suggesting that the unsubstituted and substituted
moiety plays an important role in enhancing the antibacterial ,antifungal and anti-
inflammatory properties of this class of compounds.

44
 Summary And conclusion
All the compounds showed antibacterial activity antifungal activity
against the strains tested.

 Results of the anti-inflammatory activity was found to be comparable

with the reference compound.
It indicates that this basic Thiazolidinedione (TZD) moiety can be a
potential scaffold for antibacterial,antifungal and anti-inflammatory
drugs.
 Hence, it is concluded that there is a scope of further study on
developing some lead compounds for the treatment of chronic
bacterial ,fungal infections and also many inflammatory diseases.
45
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Chem 18:2019–2028.
SPECIAL THANKS – Dr. SUBHASHINI (Dept: Chemistry,Osmania University)

48