In Vitro Evaluation of Antimicrobial Activity of

Tinospora cordifolia & Calotropis species

A THESIS
Submitted for the Award of Ph.D. Degree of
Institute of Advanced Studies in Education, Deemed to be University
(Accredited with B Grade by NAAC), Gandhi Vidya Mandir, Sardarshahar (Raj.)

Under the Supervision of

Submitted By
Dr. Gajanand Modi
Priyanka Sharma
Assistant Professor
INTRODUCTION
The steadily increasing microbial resistance to existing drugs is a serious
problem in antimicrobial therapy and necessitates continuing research into
new classes of antimicrobials (Essawi and Srour 2000; Woodford 2003).
One way to prevent antibiotic resistance of pathogenic species is to use new
compounds that are not based on existing synthetic antimicrobial agents
(Shah, 2005; Chanda et al 2011).
Plant and plant derived agents have long history to clinical relevance as a
source of potential chemotherapeutic agents (Cushnie and Lamb 2005).
There are many plants in world having many antimicrobial and antifungal
properties.
The selected plants for research are following:
(1) Calotropis Species :
(A) Calotropis procera (B) Calotropis gigantea
(2) Tinospora cordifolia
A. CALOTROPIS PROCERA
Calotropis procera of family Asclepiadaceae and Class
Dicotyledons is a tropical plant growing wild in warm climate
up to an altitude of about 1050 meters. Calotropis procera
Linn.is often found as a weed throughout India in more or
less warm dry places, predominantly in Sub -Himalayan
tracts, Deccan to Kanya- Kumari.
Figure 1:Calotropis procera
• Ecology
Calotropis procera is drought-resistant and salt-tolerant to a
relatively high degree. It often grows in saline or slightly
saline soils with low soil moisture, forming mono-specific
stands (El-Midany 2014)
• Geographical Distribution
It is native to tropical and subtropical Africa and Asia,
common in the Middle East (Parsons and Cuthbertson 2001;
Lottermoser 2011) and in Latin America, where the species
has high socioeconomic value (Abbas et al 1992).
B.CALOTROPIS GIGANTEA

Calotropis gigantea is a weed plant commonly known

as giant milk weed. The plant belongs to Apocynaceae
family which includes latex bearing plants.
Figure 2: Calotropis gigantea
• Ecology
Calotropis gigantea is drought resistant, salt tolerant to a relatively
high degree, grows wild up to 900 meters (msl) throughout the
country (Sharma and Tripathi 2009) and prefers disturbed sandy
soils with mean annual rainfall: 300-400 mm.
• Geographic Distribution
It is a native of India, China and Malaysia and distributed in the
following countries: Afghanistan, Algeria, Burkina Faso, Cameroon,
Chad, Cote d’Ivoire, Democratic Republic of Congo, Egypt, Eritrea,
Ethiopia, Gambia, Ghana, guinea-Bissau, India, Iran. Iraq, Israel,
Kenya, Kuwait, Lebanon, Libyan, Arab Jamahiriya, Mali,
Mauritania, morocco, Mozambique, Myanmar, Nepal, Niger,
Nigeria, Oman, Pakistan, Saudi Arabia.
2.TINOSPORA CORDIFOLIA
T. cordifolia, belongs to the family Menispermaceae
and is an herbaceous vine, which is distributed
throughout tropical Indian subcontinent and China, at
an elevation measuring 300 m above sea level
(Anonymous 1976).
Figure 3: Tinospora Cordifolia
• Ecology
Tinospora cordifolia prefers wide range of soil, acid to alkaline and it needs
moderate level of soil moisture. Found throughout tropical India, ascending to an
altitude of 1000 feet. It is a common plant of deciduous and dry forests, growing
over hedges and small trees (Kirti et al 2004; Bhandari 2006).

• Geographical distribution
Tinospora cordifolia is indigenous to the tropical areas of India, Myanmar and Sri
Lanka ascending to an altitude of 1200 m.
 OBJECTIVES OF INVESTIGATION
The present investigation was carried out with the leaves of these plants to
investigate the antimicrobial activities with the following objectives:

• To evaluate the in vitro antibacterial activities of the extracts of Tinospora

cordifolia
• To evaluate the in vitro antifungal activities of the extracts of Tinospora cordifolia
• To evaluate the in vitro antibacterial activities of the extracts of Calotropis species
• To evaluate the in vitro antifungal activities of the extracts of Calotropis species.
MATERIAL & METHODOLOGY
Steps
 Study Site
Steps
 Cleaning of Glass wares
 Plant Selection
 Collection of Plant material
 Choice of Solvents
Successful evaluation of antimicrobial activity of plant material is largely dependent on the type of
solvent used in the extraction procedure. Properties of a good solvent in plant extractions include:
• Low toxicity,
• Ease of evaporation at low heat,
• Promotion of rapid physiologic absorption of the extract,
• Preservative action and inability to cause the extract to complex or dissociate (Hughes 2002)
 Standard antibiotics
Different antibacterial antibiotics (Hi-Media) (Tetracyclin and Penicillin) and antifungal antibiotics
(Ketoconazole) were used in experimental work.
 Test microorganisms
Following tabulated microbes were used and these pure cultures were imported from IMTECH,
Chandigarh. Six bacterial and two fungal strains were used in study which were obtained from CSIR-
Institute of Microbial Technology, Chandigarh.
Serial
Microorganisms MTCC No.
no.
Gram Negative Bacteria
1 Escherichia coli 1692
2 Proteus vulgaris 744
3 Klebsiella pneumoniae 7407
4 Pseudomonas aeruginosa 4676
5 Enterobacter aerogenes 111
Gram Positive Bacteria
6 Staphylococcus aureus 7443
Fungal Strains
7 Trichophyton rubrum 7859
8 Epidermophyton floccosum 7880
 Maintenance of Microorganisms
The bacterial and the fungal cultures were maintained on nutrient agar medium and saborauds
dextrose agar (SDA) medium respectively. For this, selective media poured in sterilized test tubes
(15 ml). These tubes were tilted to make a slant and the media in the test tubes were allowed to
solidify. The bacterial and fungal cultures were streaking on these slants and incubated at optimum
temperature .
 Preparation of standard Microbial Culture for Experiment
 Preparation of aqueous extract
Fifty grams each of powdered leaves of T. cordifolia, C. procera and C. gigantea were macerated
with 100 ml sterile distilled water in a blender for 10 min. The macerate was first filtered through
double layered muslin cloth and centrifuged at 4000 rpm for 30 min. The supernatant was filtered
through Whatman No.1 filter paper and heat sterilized at 120°C for 30 min. The extracts were
preserved aseptically in brown bottles at 4°C until further use.
 Preparation of solvent extract
Serial exhaustive extraction which involves successive extraction with solvents of increasing
polarity from a nonpolar (petroleum ether) to a more polar solvent (Ethanol and aqueous) to ensure
that a wide polarity range of compound could be extracted will be used.
The crushed and powdered leaves (200 g) were extracted over a period of forty-eight hours in
soxhlet extraction unit successively with the solvents of increasing polarity (Petroleum ether,
Chloroform, Ethyl actate and Ethyl alcohal) to cover a wide range of compounds. Solvents were
removed in vacuum using rotary evaporation.
The evaporated extracts so obtained were preserved at 4°C in airtight bottles until further use.
 Antibacterial screening
All experiments were carried out in triplicates. Antimicrobial activity of plant extracts was
measured by the disc diffusion method and Minimum Inhibitory Concentration (MIC)
procedures.
 Procedure for performing the Disc Diffusion method
The antimicrobial screening of the extract was carried out by determining the zone of inhibition
using disc diffusion method by spread plate. The plant extracts were tested against pathogenic
bacterial strains of gram positive and Gram-negative strains and fungal strains by disc diffusion
method (Kirby and Bauer 1966).
 Inoculation of culture media
Bacterial cultures (adjusted to 1 × 106 CFU/ml using spectrophotometer) were used to lawn
Mueller-Hinton agar plates evenly using a sterile swab.
 Application of Discs to Inoculated MHA Plates
Discs approximately 5 mm in diameter were placed in laminar air flow under ultraviolet radiation
lamp for sterilization. After sterilization, discs were impregnated with 10 μl (1 mg/ml) of the plant
extracts were placed on the agar surface. Each disc was pressed down firmly to ensure complete
contact with the agar surface either with sterilized forceps. Standard antibiotic (Hi-Media) discs
were also placed on media plates (10 μg/disc). 10 μl of the respective solvent served as the negative
control.
The plates were allowed for 1 hour permitting good diffusion and then plates were then incubated at
37°C for 18-24 h . After overnight incubation the plates were examined for the zone of inhibition.
 Determination of Minimum Inhibitory Concentration
The test organisms were grown in nutrient broth medium to a concentration1 × 106 CFU/ml. Extract
of about 0.5 ml (0.25-3mg/ml) was mixed with 4 ml of nutrient broth inoculated with 0.5 ml of
bacterial suspension. The tubes containing 4.5 ml of broth and 0.5 ml of bacterial suspension served
as bacterial control and 5 ml of un-inoculated broth served as blank. The tubes were incubated at
37°C for 18 h. Inhibition of bacterial growth was determined by measuring the absorbance at 600
nm in a colorimeter. The lowest concentration of the compound that inhibits the growth of the
organism was determined as the MIC The percentage of growth inhibition was calculated according
to the formula:
Percent growth inhibition = [(Acontrol – Atest) / Acontrol] × 100
 Antifungal activity assay
The in vitro assessment of antifungal susceptibility is done by two methods: Diffusion Assay and
Broth Assay. The dermatophytes grown on SDA medium for a week were flooded with 0.85% saline.
After settling of the larger particles, conidia were counted with a hemocytometer and diluted in
saborauds dextrose broth to a final spore concentration of 1×106spores/ml. For dermatophytic assay
in broth, 5 ml of sterile saborauds dextrose broth medium taken in screw capped tubes were
inoculated with 20 μl of fungal suspension and1-10 mg/ml concentration of the extract. The tubes
were incubated for a week at 30°C. The visible mycelial growth in the tubes expressed the degree of
activity of the extract. Disc fed with corresponding solvent alone served as control.
Ketoconazole was used as a standard antifungal agent. The percentage of growth inhibition was
calculated according to the formula
Percent growth inhibition = [(Acontrol – Atest ) / Acontrol ] × 100
Statistical analysis
The experimental results are expressed as mean ± standard deviation (SD) of triplicate
measurements. The data was subjected to One Way Analysis of Variance(ANOVA).Statistical
analysis was performed using SPSS statistical software.
RESULTS AND DISCUSSIONS
The present study entitled “In vitro evaluation of Antimicrobial activity of
Tinospora cordifoia and Calotropis species” was done with two major
steps:
• Antibacterial activity assay
• Antifungal activity assay
In this study various extract of Calotropis sp. and Tinospora cordifolia
were assayed in vitro by disc diffusion method and Minimum Inhibitory
Concentration (MIC) by broth dilution technique for antimicrobial activity
against bacterial strains. Samples were extracted with 5 different solvent.
Some Gram-positive and Gram-negative pathogenic micro-organisms were
used against these extracts. Several in vitro experiments were conducted
and observed with respected to inhibition zone. Inhibition zone was
measured in mm. Standard antibiotics discs of tetracyclin (10μg) and
penicillin (10μg) were also used as standard comparison purpose.
Table 1: Zone of inhibitory activity (in millimeter) of different solvent extracts of Calotropis procera
leaves against the test bacteria
Micro-
organisms/ Escherichia Proteus Klebsiella Staphylococc Pseudomonas Enterobacter
Solvent coli vulgaris pneumoniae us aureus aeruginosa aerogenes
Extract

Aqueous 18.2±0.58 14.3±0.63 12.7±0.45 15.8±0.38 12.3±1.06 16.5±0.81

Ethanol 21.7±0.69 16.5±0.48 17.4±0.33 22.8±0.78 14.4±0.65 18.5±0.27

Chloroform 22.4±0.54 18.2±0.93 16.2±1.10 19.8±0.48 15.3±0.57 17.4±0.19

Petroleum
18.7±0.35 15.4±0.59 18.3±0.78 23.9±0.87 13.8±0.47 19.2±0.82
Ether
Ethyl
23.4±1.03 19.3±0.43 19.7±0.38 24.5±0.95 16.9±0.69 21.5±1.15
Acetate

Tetracyclin 5.6±0.37 6.5±0.85 7.0±0.46 26.8±0.78 6.3±0.66 9.2±0.49

Penicillin 7.5±0.96 6.2±0.82 8.3±0.38 30.4±0.63 7.2±0.56 6.4±0.29

Figure 4: Antibacterial activity
of ethyl acetate leaf extract of
Calotropis procera against the
test bacteria
(A) Escherichia coli
(B) Proteus vulgaris
(C) Klebsiella pneumoniae
(D) Enterobacter aerogenes
(E) Pseudomonas aeruginos
(F) Staphylococcus aureus
Table 2: Zone of inhibitory activity (in millimeter) of different solvent extracts of Calotropis gigantea leaves
against the test bacteria
Micro-
organisms/ Escherichia Proteus Klebsiella Staphylococcu Pseudomonas Enterobacter
Solvent coli vulgaris pneumoniae s aureus aeruginosa aerogenes
extract
Aqueous 12.4±0.74 13.1±0.46 10.5±0.67 15.8±0.15 14.3±0.81 11.5±1.03

Ethanol 14.5±0.65 12.1±0.57 13.3±0.76 18.9±0.33 16.8±1.10 17.5±0.46

Chloroform 16.2±0.32 14.7±0.85 12.5±1.05 17.6±0.48 17.2±0.96 13.9±0.57

Petroleum
15.4±0.24 19.6±0.73 18.3±0.98 21.5±0.85 17.5±0.62 15.8±0.43
Ether

Ethyl Acetate 16.9±1.06 21.2±0.47 19.4±0.54 23.4±0.63 21.7±0.88 18.7±0.79

Tetracyclin 5.6±0.37 6.5±0.85 7.0±0.46 26.8±0.78 6.3±0.66 9.2±0.49

Penicillin 7.5±0.96 6.2±0.82 8.3±0.38 30.4±0.63 7.2±0.56 6.4±0.29

•

Figure5: Antibacterial activity

of ethanol leaf extract of
Calotropis gigantea against the
test bacteria
(A) Escherichia coli
(B) Proteus vulgaris
(C) Klebsiella pneumoniae
(D) Staphylococcus aureus
(E) Pseudomonas aeruginosa
(F) Enterobacter aerogenes
Table 3: Zone of inhibitory activity (in millimeter) of different solvent extracts of Tinospora cordifolia leaves
against the test bacteria
Micro-
organisms/ Escherichi Proteus Klebsiella Staphylococcus Pseudomonas Enterobacter
Solvent a coli vulgaris pneumoniae aureus aeruginosa aerogenes
Extract

Aqueous 8.2±0.87 10.3±0.93 12.5±0.37 25.3±0.48 11.5±0.57 11.6±1.22

Ethanol 21.3±0.58 22.2±0.69 23.5±0.44 27.8±0.92 21.4±0.73 20.2±0.45

Chloroform 16.3±0.34 14.3±0.49 16.7±0.54 18.3±0.62 9.4±0.78 15.7±0.65

Petroleum
10.3±0.94 15.4±0.79 21.8±0.83 21.5±0.35 19.6±0.56 17.8±0.67
Ether

Ethyl Acetate 12.5±1.06 14.2±0.49 19.6±0.28 18.4±0.74 16.5±0.89 22.8±1.10

Tetracyclin 5.6±0.37 6.5±0.85 7.0±0.46 26.8±0.78 6.3±0.66 9.2±0.49

Penicillin 7.5±0.96 6.2±0.82 8.3±0.38 30.4±0.63 7.2±0.56 6.4±0.29

Figure 6: Antibacterial activity of
ethanol leaf extract of Tinospora
cordifolia against the test
bacteria
(A) Escherichia coli
(B) Proteus vulgaris
(C) Klebsiella pneumoniae
(D) Staphylococcus aureus
(E) Pseudomonas aeruginosa
(F) Enterobacter aerogenes
 Table 4: Zone of inhibitory activity (in millimeter) of different solvent extracts of Calotropis procera leaves against
Trichophyton rubrum and Epidermophyton floccosum
Petroleum
Aqueous Ethanol Chloroform Ethyl Acetate
Micro-organisms Ketonocazole Ether
Extract Extract Extract Extract
Extract
Trichophyton
14.5±0.96 8.8±0.58 14.5±1.05 13.5±0.34 17.2±0.75 19.7±0.84
rubrum
Epidermophyton
13.4±0.57 9.2±0.67 16.2±0.97 15.4±0.51 14.7±0.29 20.5±0.68
flocossum
Table 5: Zone of inhibitory activity (in millimeter) of different solvent extracts of Calotropis gigantea leaves against
Trichophyton rubrum and Epidermophyton flocossum
Ethyl
Aqueous Ethanol Chloroform Petroleum
Micro-organisms ketonocazole Acetate
Extract Extract Extract Ether Extract
Extract

Trichophyton rubrum 14.5±0.96 12.5±0.35 14.9±0.47 20.6±0.96 18.3±0.89 21.5±0.48

Epidermophyton 18.8±0.88
13.4±0.57 11.8±0.69 16.3±0.52 16.9±0.76 23.5±0.65
flocossum
Fig ure 7: Ant ifunga l act ivity of ethy l acetate extra cts of Ca lotrop is pro cera
against the test f ungi (a) Tr ic hoph yto n r ubrum (b) Ep ide rmoph yton
f lo ccosum

( a ) Tr ich ophy ton r u b r um (b) Epidermophyton floccosum

Fig ure 8: A ntifu ngal a ctivity of et hyl acetat e ext ra cts of Calo trop is
giga ntea against th e test fu ngi (a) Trichop hy ton rubr um (b) Ep ide rmoph yto n
f lo ccosum

(a) Trichophyton rubrum (b) Epidermophyton floccosum

Table 6: Zone of inhibitory activity (in millimeter) of different solvent extracts of Tinospora cordifolia
leaves against Trichophyton rubrum and Epidermophyton flocossum

Petroleum Ethyl
Aqueous Ethanol Chloroform
Micro-organisms Ketonocazole Ether Acetate
Extract Extract Extract
Extract Extract

Trichophyton
14.5±0.96 10.2±0.21 13.8±0.48 14.4±0.79 15.6±0.67 18.5±1.05
rubrum

Epidermophyton
13.4±0.57 11.3±0.98 15.9±0.58 13.6±0.38 16.3±0.89 17.4±0.75
flocossum
Fig ure 9:Ant ifunga l act ivity of ethy l acetate ext racts of Tino spora cordifo lia
a g a in st t h e t es t f u n gi ( a ) Tr ic h oph yton r u b ru m ( b ) Ep id e r mophy ton f lo c c osum

(a) Trichophyton rubrum (b) Epidermophyton floccosum

TABLES AND GRAPHS
DETERMINATION OF MIC

Broth dilution method which was used to determine the MIC values of ethyl acetate and ethanol
leaf extract of Calotropis sp. (Table 4.1.4 and Table 4.1.5) and Tinospora cordifolia (Table 4.1.6)
respectively was carried out with concentrations ranging from 0.25-3.0 mg/ml.
Minimum inhibitory concentration values for E. coli and E. aerogenes were found to be 2.25
mg/ml and P. vulgaris and S. aureus 2.50 mg/ml. But MIC values for K. pneumoniae and P.
aeruginosa were recorded 2.75 mg/ml.
At 600 nm, E. coli, P. vulgaris, K. pneumoniae, S. aureus, P. aeruginosa and E. aerogenes showed
an absorbance of 0.15 (94.98% growth inhibition), 0.21 (92.97% growth inhibition), 0.27 (90.96%
growth inhibition), 0.25 (91.63%growth inhibition), 0.3 (89.96%growth inhibition) and 0.12
(95.98% growth inhibition) as against respective control absorbance.
 Table 7 : Minimum inhibitory concentration (MIC) of Calotropis procera ethyl acetate extract using
broth dilution method
Bacterial strains
Concentration
Escherichia Proteus Klebsiella Staphylococcus Pseudomonas Enterobacter
(mg/ml)
coli vulgaris pneumoniae aureus aeruginosa aerogenes

Control 2.99±0.1 2.88±0.1 3.05±0.09 3.02±0.11 2.94±0.13 2.9±0.11

0.25 2.61±0.1 2.65±0.06 2.79±0.09 2.83±0.11 2.72±0.03 2.52±0.08
0.5 2.22±0.07 2.33±0.08 2.48±0.05 2.62±0.08 2.48±0.06 2.12±0.09
0.75 1.93±0.07 2.07±0.02 2.15±0.05 2.35±0.05 2.26±0.06 1.83±0.07
1 1.65±0.1 1.77±0.06 1.92±0.04 2.04±0.04 1.98±0.03 1.45±0.07
1.25 1.28±0.06 1.46±0.07 1.73±0.08 1.78±0.04 1.65±0.08 1.08±0.09
1.5 0.99±0.04 1.18±0.17 1.45±0.07 1.23±0.03 1.47±0.1 0.79±0.07
1.75 0.62±0.06 0.84±0.12 1.21±0.04 0.98±0.11 1.22±0.05 0.53±0.05
2 0.4±0.07 0.63±0.08 0.86±0.09 0.7±0.05 0.95±0.09 0.32±0.05
2.25 0.15±0.04 0.45±0.03 0.67±0.1 0.47±0.07 0.72±0.11 0.12±0.02
2.5 0 0.21±0.04 0.45±0.05 0.25±0.07 0.49±0.03 0
2.75 0 0 0.27±0.05 0 0.3±0.04 0
3 0 0 0 0 0 0
Figure 10 : Per cent growth inhibition of bacteria at different conc. (0.25- 3.0 mg/ml) of Calotropis
procera ethanol leaf extract
Table 8: Minimum inhibitory concentration (MIC) of Calotropis gigantea ethyl acetate extracts using
broth dilution method
Bacterial strains
Concentration
Escherichia Proteus Klebsiella Staphylococcus Pseudomonas Enterobacter
(mg/ml)
coli pneumoniae aureus aeruginosa aerogenes
Vulgaris
Control 2.99±0.08 2.88±0.09 3.05±0.08 3.02±0.08 2.94±0.04 2.9±0.09
0.25 2.83±0.11 2.52±0.09 2.63±0.11 2.47±0.07 2.79±0.08 2.74±0.07
0.5 2.64±0.09 2.33±0.07 2.42±0.03 2.26±0.1 2.5±0.1 2.46±0.08
0.75 2.39±0.07 2.1±0.07 2.21±0.02 1.92±0.06 2.26±0.05 2.08±0.03
1 2.12±0.07 1.97±0.07 2.02±0.04 1.53±0.06 1.94±0.06 1.72±0.09
1.25 1.93±0.1 1.45±0.06 1.85±0.08 1.21±0.06 1.63±0.06 1.41±0.09
1.5 1.54±0.08 1.08±0.04 1.37±0.05 0.95±0.03 1.3±0.08 1.12±0.12
1.75 1.29±0.06 0.75±0.06 0.84±0.12 0.62±0.04 0.97±0.04 0.79±0.04
2 0.82±0.11 0.39±0.06 0.45±0.04 0.31±0.07 0.66±0.1 0.54±0.06
2.25 0.36±0.09 0.15±0.06 0 0 0.46±0.08 0.42±0.01
2.5 0 0 0 0 0.27±0.07 0.31±0.03
2.75 0 0 0 0 0.11±0.04 0.14±0.05
3 0 0 0 0 0 0
Figure 11 : Per cent growth inhibition of bacteria at different conc. (0.25- 3.0 mg/ml) of Calotropis
gigantea ethanol leaf extract
Table 9: Minimum inhibitory concentration (MIC) of Tinospora cordifolia ethanol extract using broth
dilution method
Bacterial strains
Concentration
Escherichia Proteus Klebsiella Staphylococcus Pseudomonas Enterobacter
(mg/ml)
coli vulgaris pneumoniae aureus aeruginosa aerogenes
Control 2.99±0.11 2.88±0.12 3.05±0.09 3.02±0.06 2.94±0.09 2.9±0.07
0.25 2.54±0.09 2.31±0.1 2.56±0.1 2.19±0.03 2.22±0.12 2.55±0.1
0.5 2.31±0.06 2.12±0.03 2.39±0.09 1.9±0.07 2.05±0.06 2.34±0.13
0.75 2.02±0.05 1.94±0.04 2.09±0.09 1.61±0.05 1.92±0.05 2.02±0.05
1 1.89±0.08 1.78±0.06 1.85±0.06 1.25±0.05 1.7±0.03 1.85±0.06
1.25 1.54±0.08 1.59±0.09 1.5±0.08 1.18±0.06 1.61±0.07 1.59±0.04
1.5 1.3±0.09 1.26±0.04 1.39±0.06 1±0.05 1.39±0.08 1.42±0.09
1.75 1.16±0.07 1.02±0.07 1.2±0.08 0.72±0.06 1.22±0.07 1.36±0.07
2 0.92±0.12 0.87±0.07 0.95±0.08 0.5±0.05 1±0.04 1.24±0.12
2.25 0.69±0.05 0.64±0.09 0.71±0.12 0.26±0.05 0.77±0.12 0.98±0.11
2.5 0.38±0.07 0.37±0.07 0.55±0.07 0 0.49±0.1 0.73±0.13
2.75 0.12±0.03 0.13±0.03 0.31±0.08 0 0.22±0.03 0.49±0.05
3 0 0 0.22±0.03 0 0 0.2±0.03
Figure 12 : Per cent growth inhibition of bacteria at different conc. (0.25- 3.0 mg/ml) of Tinospora
cordifolia ethanol leaf extract
Table 10: Minimum inhibitory concentration (MIC) of Calotropis procera ethyl acetate extract
using broth dilution method
Concentration (mg/ml) Fungal strains
Trichophyton rubrum Epidermophyton floccosum
Control 2.52±0.1 2.73±0.08
1 2.05±0.06 2.25±0.07
2 1.9±0.08 2.07±0.08
3 1.65±0.08 1.91±0.12
4 1.38±0.1 1.6±0.17
5 1.1±0.05 1.22±0.05
6 0.67±0.1 0.98±0.09
7 0.3±0.05 0.6±0.07
8 0.11±0.03 0.26±0.06
9 0 0
10 0 0
11 0
Figure 13 : Percent growth inhibition of bacteria at different concentrations (1-10 mg/ml) of
Calotropis procera ethyl acetate leaf extract
Table 11: Minimum inhibitory concentration (MIC) of Calotropis gigantea\ ethyl acetate extract using
broth dilution method
Concentration (mg/ml) Fungal strains
Trichophyton rubrum Epidermophyton floccosum

Control 2.52±0.06 2.73±0.07

1 1.85±0.15 2.18±0.13
2 1.62±0.07 1.99±0.14
3 1.4±0.05 1.64±0.08
4 1.21±0.08 1.33±0.08
5 1.08±0.08 1.12±0.04
6 0.85±0.05 0.93±0.11
7 0.53±0.04 0.75±0.09
8 0.22±0.06 0.43±0.07
9 0 0.2±0.06
10 0 0
11 0 0
12
0 0
Figure 14: Percent growth inhibition of bacteria at different concentrations (1-10 mg/ml) of Calotropis
gigantea ethyl acetate leaf extract
Table 12 : Minimum inhibitory concentration (MIC) of Tinospora cordifolia ethyl acetate extract
using broth dilution method

Concentration (mg/ml) Fungal strains

Trichophyton rubrum Epidermophyton floccosum

Control 2.52±0.09 2.73±0.05

1 2.15±0.12 2.08±0.05
2 2.08±0.07 1.88±0.1
3 1.75±0.08 1.57±0.09
4 1.52±0.1 1.42±0.09
5 1.26±0.04 1.2±0.09
6 0.97±0.1 0.98±0.06
7 0.62±0.12 0.72±0.07
8 0.34±0.05 0.47±0.11
9 0.07±0.03 0.3±0.06
10 0 0
Figure 15 : Percent growth inhibition of bacteria at different concentrations (1-10 mg/ml) of
Tinospora cordifolia ethyl acetate leaf extract
 Analysis of variance ethyl acetate extract of three plants against Staphylococcus aureus
SUMMARY
Groups Count Sum Average Variance
T. cordifolia 13 14.82 1.14 0.872983
C. procera 13 18.27 1.405385 1.201377
C. gigantea 13 14.39 1.106923 1.107056

ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 0.695944 2 0.347972 0.328129 0.722402 3.259446
Within Groups 38.177 36 1.060472
Total 38.87294 38
Analysis of variance ethanol extract of three plants against Staphylococcus aureus
SUMMARY
Groups Count Sum Average Variance
T. cordifolia 13 13.63 1.048462 0.884914
C . procera 13 12.89 0.991538 0.863447
C . gigantea 13 15.74 1.210769 1.114624

ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 0.336467 2 0.168233 0.176284 0.839099 3.259446
Within Groups 34.35583 36 0.954329
Analysis of variance ethyl acetate extract of three plants against Trichophyton rubrum
SUMMARY
Groups Count Sum Average Variance
T. Cordifolia 13 13.28 1.021538 0.844964
C. Procera 13 11.68 0.898462 0.838064
C. gigantea 13 11.28 0.867692 0.687669

ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 0.172308 2 0.086154 0.109023 0.897004 3.259446
Within Groups 28.44837 36 0.790232
Total 28.62068 38
Analysis of variance ethyl acetate extract of three plants against Epidermophyton floccosum
SUMMARY
Groups Count Sum Average Variance
T. Cordifolia 13 13.35 1.026923 0.776273
C. Procera 13 13.62 1.047692 0.967936
C. Gigantea 13 13.3 1.023077 0.826973

ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 0.004559 2 0.002279 0.00266 0.997344 3.259446
Within Groups 30.85418 36 0.857061
Total 30.85874 38
OBSERVATIONS
 In vitro antibacterial activity studies Antimicrobial Activity
• From the results obtained, it was found that ethyl acetate extract of Calotropis sp and ethanol extract of
Tinospora cordifolia was the best solvent for extracting antimicrobial compounds from leaves in all the three
plants. Ethyl acetate leaf extract of Calotropis procera and Calotropis gigantea showed higher inhibition
zone against all bacterial species while ethanol leaf extract of Tinospora cordifolia showed maximum
inhibition zone.

• S. aureus showed maximum (24.5 mm) inhibition zone in ethyl acetate extract while the aqueous extract
which showed an inhibition of zone 12.3mm against P. aeruginosa.

• The highest antibacterial activity of Calotropis gigantea as indicated by the zone of inhibition was achieved
with ethyl acetate extract which showed inhibition zones of 16.9 mm, 21.2 mm, 19.4 mm, 23.4 mm, 21.7
mm and 18.7 mm against E. coli, P. vulgaris, K. pneumoniae, S. aureus, P. aeruginosa, E. aerogenes
respectively. Among the bacteria tested, S. aureus showed maximum susceptibility to C.gigantea ethyl
acetate extract. Aqueous extracts of C. gigantea showed the least antibacterial activity.

• Among the bacteria tested, S. aureus showed maximum susceptibility to T. cordifolia ethanol extract.
Chloroform and aqueous extracts showed the least antibacterial activity.

• In the case of standard antibiotics (Tetracycline and Penicillin) maximum inhibition zone was reported
against S. aureus in both Tetracyclin (26.8 mm) & Penicillin (30.4mm). Minimum inhibition zone was
obtained against E. coli (5.6 mm) in Tetracyclin.
 In vitro antifungal activity studies

• Among the solvent extracts tested, ethyl acetate extract had a broad spectrum of activity against both fungi
tested and showed the highest zones of inhibition against E. flocossum (20.5 mm). The least zone of
inhibition was observed with the aqueous extract which showed an inhibition of zone 8.8mm against T.
rubrum.

• The highest antifungal activity of C. gigantea as indicated by the zone of inhibition was achieved with
ethyl acetate extract which showed inhibition zones of 21.5mm and 23.5mm against T.rubrum E. floccosum
respectively.

• T. cordifolia leaf extracts revealed the highest antifungal activity with ethyl acetate extract which showed
inhibition zones of 18.5 mm and 17.4 mm against T. rubrum and E. flocossum respectively. Both fungal
strains showed susceptibility to T. cordifolia all the extract. Aqueous extracts showed the less antifungal
activity.

• MIC values were evaluated for ethyl acetate extract for all three plants species concentration ranging 1-10
mg/ml. Both dermatophytes, T.rubrum and E. floccosum were found to be more susceptible to the ethyl
acetate leaf extract of all the three plants. Minimum inhibitory concentration values for ethyl acetate extract
of C.procera against T.rubrum and E. aerogenes were found to be 8 mg/ml.