Antimicrobial activity of two Indian

medicinal plants ​Tinospora cordifolia

(Family:Menispermaceae) and ​Cassia fistula
(Family: Caesalpiniaceae) against human
pathogenic bacteria

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CONTENTS

S.NO TITLE PAGE.NO

1 ABSTRACT
4

2 INTRODUCTION 5 - 24

3 BACTERIA AND FUNGI 25 – 40

4 LITERATURE REVIEW 41- 42

5
DRUG PROFILE 43– 47

5 PLANT PROFILE 48 – 52

6 AIM AND OBJECTIVE 53 – 54

7 PLAN OF WORK 55
8
METHOD AND METHODOLOGY 56 - 60

9 PHYTOCHEMICAL SCREENING TEST 61- 64

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10
ESTIMATION OF ANTIBACTERIAL AND 65– 66
ANTIFUNGAL ACTIVITY

11 RESULTS AND DISCUSSION 67 - 79

12 CONCLUSION 80

13 BIBLIOGRAPHY 81 - 95

LIST OF ABBREVIATION

% : percentage

Ml : milliliter

µg : microgram

G : gram

Kg : Kilogram

Min : minute

Sec : seconds

W/v : weight / volume

V/v : volume / volume

Fig : figure

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conc. : Concentrated

ppt : precipitated

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ABSTRACT​:

In the present investigation antimicrobial activity of two Indian medicinal plants i.e. ​Tinospora
cordifolia a​ nd ​Cassia fistula w ​ ere evaluated against seven human pathogenic bacterial strains.
Both were tested by serial micro-dilution method. Antimicrobial susceptibility of aqueous and
solvent extracts was assessed. For this purpose, both positive and negative controls were set to
determine MIC and MBC values. After bioassays, aqueous and solvent extracts of ​Tinospora
cordifolia a​ nd ​Cassia fistula e​ xhibited significant antimicrobial susceptibility against bacteria
both Gram negative and Gram positive i.e. ​Klebsiella pneumoniae​, ​Escherichia coli,
Micrococcus luteus, Streptococcus pneumoniae,Staphylococcus aureus, Bacillus​ ​cereus and
Lactobacillus acidophilus at a very low concentration. More specifically, higher percent growth
inhibition was obtained in the presence of aqueous extracts in comparison to solvent extracts,
which was much higher than synthetic antibiotics. Further, different extracts have shown very
low MIC
value, which was obtained in a range of 0.0078-0.125 mg/ml. It was much lower than the MIC
values (0.223-0.892 mg/ml) obtained in presence of standard antibiotics i.e. tetracycline,
ampicillin and ciprofloxacin. More specifically, aqueous extracts of both plants have shown
lower MIC (0.00975 mg/ml in ​E. coli and K.​ ​pneumoniae)​ ​and ​MBC values (0.078 mg/ml in ​E.
coli a​ nd ​K. pneumoniae)​ than broad-spectrum antibiotics (0.223-0.892 mg/ml). Further, aqueous
extracts of both plant species have shown significantly much higher inhibition zone diameters
(20-30mm) against all seven bacterial strains than the synthetic antibiotics (6-18). Certainly,
above antimicrobial effects are attributed due to presence of certain chemical substances in the
leaves and legumes of above plant species.

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 ​ ​INTRODUCTION
In the present time multiple drug resistance in microbial pathogens became a serious health probl
em to humankind worldwide.It is aroused due to indiscriminate and repetitive use of
antimicrobial drugs by inadequate disease treatment. To acquire drug resistance microbes have
developed a new enzyme system to cleave the drug and make it useless for control of infection.
Hence, plant origin herbal medicines are considered as safe alternatives of synthetic drugs. There
are varied methods of medicines like Ayurveda, Homeopathy and Unani, which utilize plant
materials for drug production. Currently, Ayurveda is considered as a vital system of medicine
and governs the worldwide recognition and having non-toxic substances.

Hence, in the last few decades, so many plant species were explored for obtaining
potential antimicrobials for therapeutic purposes, which later on became an integral part of
primary health care in many parts of the world. Hence, plants, which possess strong
antimicrobial potential against pathogens are considered as valuable sources of medicinal
compounds and show lesser side effects. Plants are a rich source of a wide variety of secondary
metabolites viz. tannins, terpenoids, alkaloids, and flavonoids, which possess enormous
antimicrobial properties. Approximately 25 to 50 % of current pharmaceuticals are derived from
plants. Most of them were found effective against many pathogenic bacteria, fungi, viruses and
​ esides this, few antimicrobials such
even found active against drug-resistant microorganisms​. B
as essential oils, plant extracts and pure compounds have shown broad-spectrum antimicrobial
activity against pathogens. Some of the plant products are used as nutraceuticals and in
food preservation. In the present investigation, two indigenous plant species from India have
been screened for their antimicrobial activities. ​Tinospora cordifolia i​ s a large glabrous
ascending shrub belonging to a family Menispermaceae and Caesalpiniaceae.

AIM AND OBJECTIVE

Antibiotics are one of our most important weapons in fighting bacterial infections and have
greatly benefited the health-related quality of human life since their introduction. However, over
the past few decades, these health benefits are under threat as many commonly used antibiotics
have become less and less effective against certain illnesses not only because many of them
produce toxic reactions, but also due to emergence of drug-resistant bacteria. It is essential to
investigate newer drugs with lesser resistance. Drugs derived from natural sources play a
significant role in the prevention and treatment of human diseases. In many developing
countries, traditional medicine is one of the primary healthcare systems. Herbs are widely
exploited in traditional medicine and their curative potentials are well documented. About 61%
of new drugs developed between 1981 and 2002 were based on natural products and they have
been very successful, especially in the areas of infectious disease and cancer. Recent trends,

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however, show that the discovery rate of active novel chemical entities is declining. Natural
products of higher plants may give a new source of antimicrobial agents with possibly novel
mechanisms of action. The effects of plant extracts on bacteria have been studied by a very large
number of researchers in different parts of the world. Much work has been done on ethno
medicinal plants in India.

In the current investigation carried out, a screening of ethanolic extracts of ​T.cordifolia

and ​Cassia fistula against pathogenic bacteria and fungi are done in order to detect new sources
of antimicrobial agents.

Plants are rich in a wide variety of secondary metabolites such as tannins, terpenoids,
alkaloids, flavonoids, glycosides, etc., which have been found in vitro to have antimicrobial
properties.

Herbal medicines have been known to man for centuries. Therapeutic efficiency of many
indigenous plants for several disorders has been described by practitioners of traditional
medicine. Antimicrobial properties of medicinal plants are being increasingly reported from
different parts of the world. The World Health Organization estimates that plant extracts or their
active constituents are used as folk medicine in traditional therapies of 80% of the world's
population. The harmful microorganisms can be controlled with drugs and these results in the
emergence of multiple drug-resistant bacteria and it has created alarming clinical situations in the
treatment of infections. The pharmacological industries have produced a number of new
antibiotics; resistance to these drugs by microorganisms has increased. In general, bacteria have
the genetic ability to transmit and acquire resistance to synthetic drugs which are utilized as
therapeutic agents.

MATERIALS AND METHODOLOGY

PLAN OF WORK

1. Collection and authentication.

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 2. Extraction of the plant material using reflux condensation method.
3. Preliminary phytochemical tests of ethanolic extract to identify the phytoconstituents
4. And finally estimation of antimicrobial and antifungal activity
COLLECTION AND AUTHENTICATION OF PLANT MATERIAL

The plant material Tinospora cordifolia and ​Cassia fistula ​were collected in the month of MAY
-2019 from Pushkaravanam, Rajamahendravaram​.

PREPARATION OF EXTRACTS : Plant parts of ​Tinospora cordifolia a​ nd ​Cassia fistula

were collected from forest areas of Gorakhpur in eastern U.P. The stem of ​Tinospora cordifolia
and legumes of ​Cassia fistula ​were chopped into small pieces, dried, milled and powdered by
pestle and mortar.the acetone,methanol,chloroform and petroleum ether extracts were prepared
by using 50g of dried powder of​ C.fistula​ legumes and leaves of ​Tinospora cordifolia​ in 250ml
of solvent separately.Each solvent extract was fractionated to get different serial fractions in
different solvent. The extract was dried;residue was weighed and dissolved in a known quantity
of fresh solvent.In the preparation of aqueous extract ,50mg of powdered material was dissolved
in 250ml of distilled water and kept for solubilization overnight at room temperature. For
quantization plant extracts were evaporated and redissolved in the known volume of each solvent
separately.
MICRO-ORGANISMS
Cultures of seven pathogenic bacteria strains each of Escherichia coli, Bacillus
cereus,Lactobacillus acidophilus,Micrococcus luteus,Staphylococcus aureus,Klebsiella
pneumoniae and Streptococcus pneumoniae were maintained in the laboratory in Luria broth
regularly for four days at 37 degree centigrade before use in experiments. For experiments a
portion of the overnight culture was mixed in the tests and control for inoculation.For activity
testing bacterial cultures were stored at 4 degree centigrade and subcultured after every eighth
day in solid agar plates.

DETERMINATION OF MIC AND MBC VALUES

For determination of antimicrobial activity, bacterial growth inhibition was accessed in the
presence of different increasing concentrations of ​Tinospora cordifolia ​and ​Cassia fistula
extracts. For this purpose both crude extracts were separately​ d​ iluted by using a serial micro
dilution method with Luria Broth culture medium at a​ ​final concentration range from 8.0 to
0.00975 mg/ml. The tested crude extracts and pure compounds were added to fresh suspension
after following the serial dilutions up to 10 to the power of -10. Each extract was assayed in
triplicate. Before conducting experiments all the conditions for ​in vitro ​antimicrobial activity
were standardized to determine MIC and MBC values. The MIC values are considered as the
lowest concentration of crude extract and pure compounds, which have shown no turbidity in the
culture flask after 24 hrs of incubation at 37 degree Centigrade.The turbidity in the culture flasks
was considered as visible growth of microorganisms. Further, it was standardized in terms of
absorbance at 600 nm in a visible spectrophotometer. For determination of minimum bactericidal

Page ​8
concentration (MBC) growth inhibitory assays were performed. For this purpose inoculum size
was adjusted to prepare a final colony number as 108 colony forming units (CFU/ml) in sterile
agar plates.
The incubation of test and control cultures was performed at 37degree centigrade for
24 hours. The least concentration at which no visible growth was obtained in agar plates was
considered as MBC value. For evaluation of inhibition, both negative (with antibiotics) and
positive (without antibiotics and extracts) parallel controls were set for each and every test.
Bacterial growth was obtained in presence and absence of various concentrations of solvent and
aqueous extracts of ​T. cordifolia​ and ​Cassia fistula ​separately.

MEASUREMENT OF INHIBITION ZONE DIAMETER

Antimicrobial susceptibility of ​Tinospora cordifolia ​and ​Cassia fistula ​extracts was evaluated by
agar disc diffusion method of Kirby ​et al., (​ 1966). Molten agar was used as a media for bacteria.
For this purpose six different concentrations of each extract and pure compounds were coated on
sterile filter paper discs (Whatman No. 1) of 6 mm in sizes. Extract coated discs were dried
under a laminar flow cabinet. Before experiment inoculum size was determined and adjusted to
prepare a final colony number as 108 CFU/ml (Colony Forming Unit) in sterile agar
plates.Bacterial inoculum was spread evenly on to the surface of agar plate with sterile rubber
pad spreader before the coated discs were positioned on the inoculated agar surface. Each extract
and pure compound was assayed in triplicate. For comparison, three broad spectrum antibiotics
i.e. tetracycline, ampicillin and ciprofloxacin were also used parallel as controls. All treated and
untreated plates were incubated for 24 hrs at 37 degrees centigrade. The antibacterial activity
was assessed and the size of inhibition zone diameter surrounding the filter paper discs was
measured.

MEDIA PREPARATION AND ITS STERILIZATION

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For antimicrobial susceptibility testing both solid (Agar-agar) and liquid agar
(Luria broth) media were used. It has shown a good bacterial growth and
reproducibility.
For suspension culture of bacterial cells 1% Luria Broth (w/v) was prepared by
dissolving 1 gm of broth media in 100 ml of distilled water, while 2% agar solid
was used for developing surface colony growth.

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 PHYTOCHEMICAL SCREENING

PHYTOCHEMICAL SCREENING

TEST FOR GLYCOSIDES

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 A portion of the extract was hydrolyzed with HCl and the hydrolysate was
subjected to Legal and Born Trager’s test to detect the presence of different
Glycosides.

LEGAL’S TEST

To extract, 1ml of Pyridine and a few drops of Sodium nitroprusside were added
and it was made alkaline with NaOH. Appearance of pink to red colour shows the
presence of glycosides.

BORNTRAGER’S TEST

Extract was treated with Chloroform and then the chloroform layer was separated.
To this equal quantity of dilute Ammonia solution was added. Ammonia layer
acquires pink colour showing the presence of Glycosides.

TEST FOR SAPONINS

FROTH TEST

Place 2ml of extract in water in a test tube. Shake well, stable froth (Foam)is
formed.

Hence saponins are present.

TEST FOR ALKALOIDS

DRAGENDORFF’S REAGENT

Drug extract when treated with Potassium Bismuth Iodide Solution gives reddish
brown ppt.

Hence Alkaloids are present.

MAYER’S REAGENT

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Drug extract when treated with Potassium Mercuric Iodide solution gives cream
color ppt.

Hence alkaloids are Present.

TEST FOR TANNINS

To extract a few ml of Chromic acid was added. No ppt was found.

Hence Tannins are present.

TEST FOR FLAVONOIDS

SHINODA TEST

To the extract add a few Magnesium turnings and con HCl dropwise. Pink scarlet,
crimson red or occasionally green to blue colour appears after a few mins.

Hence Flavonoids are present​.

ZINC HYDROCHLORIDE TEST

To the extract add a mixture of Zn dust and con HCl. Gives red colour after a few
min.

Hence Flavonoids are present.

TEST FOR MUCILAGE

To the extract Ruthenium red solution is added, pink colour is obtained.

Hence mucilage is present.

TEST FOR CARBOHYDRATES

MOLISCH TEST

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To the extract add a few drops of alcoholic α-naphthol, and then add a few drops of
con. H​2​SO​4 through
​ sides of the test tube, violet colour ring is appeared at the
junction.

Hence Carbohydrates are present.

TEST FOR PROTEINS

XANTHOPROTEIC TEST

To 5ml of extract add 1ml of conc Nitric acid and boil. Yellow ppt was obtained.
After cooling add 40% NAOH solution. Orange colour was obtained.

Hence proteins are present.

TEST FOR PHYTOSTEROLS

SALKOWSKI TEST

To the extract add few drops of conc H​2​SO​4, re colour at the lower layer indicate
the presence of sterol and yellow colour presence indicate

Hence Phytosterols are present.

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ESTIMATION OF ANTIBACTERIAL ACTIVITY:

MEDIA:

Soybean casein digest agar media

Sabouraud dextrose agar

METHOD OF PREPARATION​:

1​. PREPARATION OF MEDIA FOR BACTERIAL​:

40 gms of soya bean casein digest media was added to 1000 ml of purified water in
a conical flask. Then the media was subjected to sterilization in autoclave for 121​o
c for 15 min.

2. ​METHODS

A​. Estimation of TBC​:

Take 5 Petri plates in which 2 Petri plates act as a test for antibacterial, 1 petri plate
acts as standard ampicillin, 1 petri plate acts as blank and 1 petri plate for control.

Add 1ml of test sample media into the petri plate (which was prepared for total
bacteria count) and then it was incubated at 22°c for 5 days. After incubation the
zone of inhibition was studied in the petri plates.

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B​.​ Estimation of TFC

Take 5 Petri plates in which 2 Petri plates was act as test extracts, 1 petri plate acts
as blank, 1petriplate for nystatin standard and 1 petri plates for control

Add 1ml of test sample media into the petri plate (which was prepared for total
fungal count) and then it was incubated at 32​0​c for 7days. After incubation the zone
of inhibition was studied in the petri plates.

S.n Media technique Sample Pathogen No.of plates Temperature Incubation

o type Induced Blank test °c period
(test)
1 SCDA Cup-plate 1ml each E.coli 2 30-35 24-72 hrs.
2 SCDA Cup-plate 1ml each Bacillus 2 30-35 24-72 hrs.
3 SCDA Cup-plate 1ml each Salmonella 1 2 30-35 24-72 hrs.
4 SCD Cup-plate 1ml each Staphyloco 2 30-35 24-72hrs
ccus
5 SDA Cup-plate 1ml each Candida 2 32​0​C 5days
6 SDA Cup-plate 1ml each Aspergillus 1 2 32​0​C 5 days

7 SCDA Cup-plate 1ml each Ampicillin 1 22​0​C 24-72hrs

(Std. for
antibacteria
l

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8 SDA Cup-plate 1ml each Nystatin 1 32​0​C 5 days
(std. for
antifungal)

​RESULTS AND DISCUSSION

Table:

PERCENTAGE YIELD OF THE EXTRACT:

S.No Name of The Plant Percentage Yield (%)

1 T.cordifolia 12.3 %
2 Cassia fistula 9.8 %

PHYTOCHEMICAL SCREENING​:

Table:

s.no Name of the plant Alk Carb Gly Tan Phytos Flav sapo Pro muci
1 T.cordifolia + + - + + + + - +
2 Cassia fistula - + - + + + + + +

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 The above table indicates the presence (+) or absence (-) of phytochemicals in ethanolic
extract (Alk: Alkaloids, Carb: Carbohydrates, Gly: Glycosides, Tan: Tannins, Photos:
Phytosterol, Flav: Flavonoids, Sapo: Saponins , Pro: Proteins , Muci: Mucilage)

Table 1. MIC values obtained in presence of solvent and aqueous extracts of ​Tinospora cordifolia ​and ​Cassia fistula a​ gainst seven
pathogenic bacte- rial strains (mg/ml).
Table 3. Inhibition zone diameters obtained in presence of different solvent and aqueous extracts of ​Tinospora cordifolia a​ nd ​Cassia
​ ere measured by agar disc diffusion method (in mm) and compared with broad spectrum antibiotic drugs.
fistula w

Tinospora cordifolia

Plant extracts ​ K.pneumoniae E.coli B.cereus M.luteus L.acidophilus S.aureus S.pneumoniae

Chloroform 0.0585 0.0146 0.2343 0.0585 0.0292 0.0292 0.0585

Acetone 0.0625 0.0312 0.0312 0.0156 0.0625 0.0156 0.125

Methanol 0.0625 0.125 0.0312 0.0156 0.0625 0.0625 0.0156

Aqueous 0.0195 0.00975 0.039 0.0195 0.039 0.039 0.0195

Cassia fistula

Chloroform 0.0078 0.0156 0.0312 0.0156 0.0312 0.0312 0.0156

Acetone 0.0156 0.0156 0.0156 0.0312 0.0312 0.0312 0.0156

Methanol 0.0625 0.0312 0.0156 0.0156 0.0156 0.0156 0.0156

Pet.ether 0.0312 0.0625 0.0312 0.0156 0.0078 0.125 0.0078

Aqueous 0.0975 0.048 0.048 0.048 0.048 0.024 0.024

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Antibiotics

Tetracycline* 0.446 0.446 0.446 0.223 0.446 0.446 0.223

Ampicillin* 0.223 0.446 0.446 0.892 0.223 0.446 0.223

Ciprofloxacin*0.892 0.446 0.892 0.223 0.223 0.446 0.223

MIC-​Minimum Inhibitory Concentration.

Solubility of each plant extract was determined before treatment. No turbidity was found visible
after 24 hr incubation. Antibiotics were used for comparison.

Table 2 . MBC values obtained in presence of solvent and aqueous ex- tracts of ​Tinospora cordifolia a​ nd ​Cassia fistula
against seven pathogenic bacterial strains (mg/ml).

Tinospora cordifolia

Plant extracts ​ K.pneumoniae E.coli B.cereus M.luteus L.acidophilus S.aureus S.pneumoniae

Chloroform 0.1171 0.0292 0.4687 0.1171 0.0585 0.1171 0.2343

Acetone 0.125 0.25 0.125 0.0625 0.125 0.125 0.25

Methanol 0.25 0.25 0.0625 0.0312 0.125 0.125 0.0312

Pet.ether 0.125 0.25 0.0625 0.25 0.0625 0.25 0.125

Aqueous 0.078 0.39 0.078 0.078 0.078 0.078 0.078

Cassia fistula

Chloroform 0.0312 0.0312 0.0625 0.0312 0.0625 0.0625 0.125

Acetone 0.0625 0.0625 0.0625 0.0625 0.0625 0.0625 0.0312

Methanol 0.25 0.125 0.0312 0.0625 0.0625 0.0625 0.0625

Pet.ether 0.0625 0.125 0.0625 0.0312 0.0312 0.25 0.0156

Aqueous 0.0975 0.048 0.048 0.048 0.048 0.024 0.024

Antibiotics

Tetracycline* 0.892 0.892 1.78 3.57 0.892 0.892 0.892

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Ampicillin* 1.78 1.78 1.78 7.14 0.892 0.892 0.892

Ciprofloxacin*1.78 1.78 1.78 0.892 3.57 0.892 0.892

MBC-​Minimum Bactericidal Concentration

3. ​Antimicrobial activity

Images: order: staphylococcus, bacillus, E.coli, salmonella of ​T.cordifolia

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Order: staphylococcus, bacillus, E.coli, Salmonella of ​Cassia fistula

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A and C are Aspergillus brasiliensis and candida albicans of ​T.cordifolia

​ B and D are Aspergillus brasiliensis and candida albicans of ​Cassia fistula

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 Estimation of antimicrobial activity for E.coli
s.no Name of extract Zone of inhibition (mm)
1 T.cordifolia 3.4
2 Cassia fistula 4.6
3 Ampicillin 5.1

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 Estimation of antimicrobial activity for Staphylococcus aureus
s.no Name of extract Zone of inhibition (mm)
1 T.cordifolia 3.7
2 Cassia fistula 4.6
3 Ampicillin 5.1

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 Estimation of antimicrobial activity for Bacillus subtilis
s.no Name of extract Zone of inhibition (mm)
1 T.cordifolia 2.2
2 Cassia fistula 3.9
3 Ampicillin 5.8

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 Estimation of antimicrobial activity for Salmonella
s.no Name of extract Zone of inhibition (mm)
1 T.cordifolia 1.3
2 Cassia fistula 5.1
3 Ampicillin 5.6

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 Estimation of antifungal activity for Aspergillus brasiliensis
s.no Name of extract Zone of inhibition (mm)
1 T.cordifolia 4.8
2 Cassia fistula 5.1
3 Nystatin 6.2

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 Estimation of antifungal activity for Candida albicans
s.no Name of extract Zone of inhibition (mm)
1 T.cordifolia 3.8
2 Cassia fistula 2.6
3 Nystatin 6.2

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 Percentage inhibition of bacterial growth
s.no Name of bacteria % inhibition by Musa % inhibition by Mangifera
acuminata indica
1 Staphylococcus aureus 72.54 90.19
2 Bacillus subtilis 37.93 67.24
3 Escherichia coli 66.66 90.19
4 salmonella 23.21 91.07

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 Percentage inhibition of fungal growth
s.no Name of bacteria % inhibition by Musa % inhibition by Mangifera
acuminata indica
1 Aspergillus brasiliensis 77.41 82.25
2 Candida albicans 61.29 41.93

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 RESULTS​:

DETERMINATION OF MIC AND MBC VALUES:

​ hloroform extract of ​T. cordifolia s​ howed least MIC value i. e. o.146 mg/ml against ​E. coli ​while acetone
C
extract 0.0156 mg/ml against ​S. pneumoniae​. Aqueous extract of ​T. cordifolia ​showed lowest MIC values
i.e. 0.00975 mg/ml against ​E. coli, w​ hile methanol extract has shown highest activity against ​M. luteus ​and
S. pneumoniae ​at 0.0156 mg/ml concentration (Table 1)​. P ​ etroleum ether extract of ​T. cordifolia s​ howed
high activity against ​E. coli, B. cereus, a​ nd ​L. acidophilus ​at 0.312 mg/ml (Table 1)

Similarly, ​Cassia fistula ​chloroform extract was found to be highly susceptible, as it has shown very low
MIC value i.e. 0.0078 mg/ml against ​K. pneumoniae. ​Acetone extract has shown 0.0156 mg/ml MIC value
against ​K. pneumoniae, E. coli, B. cereus a​ nd ​S. pneumoniae.​ ​Cassia fistula ​aqueous extract has shown MIC
value i.e. 0.0975 mg/ml in ​K. pneumoniae a​ nd 0.048 mg/ml in ​E. coli, B. cereus,​ ​M. luteus a​ nd ​L.
acidophilus.​ Besides this, antibiotics such as tetracycline, ampicillin and ciprofloxacin have shown higher
MIC values which were obtained in a range of 0.892-3.57mg/ml (Table 1).

Lowest MBC value was found in ​Tinospora cordifolia ​methanol extract i.e. 0.0312 mg/ml in ​M. luteus ​and
S. pneumoniae ​(Table 2). Similarly, ​Cassia fistula e​ ther extract has shown very low MBC values i.e. 0.0156
mg/ml in ​S. pneumoniae (​ Table 2), while chloroform extract has shown MBC values i.e. 0.0312 mg/ml
MBC value against ​K. pneumoniae, E. coli a​ nd ​M. luteus​, while it’s aqueous extract has shown very low
MBC values i.e. 0.39 mg/ml in ​K. pneumoniae, E. coli, S. aureus a​ nd ​B. cereus a​ lso than antibiotics.
Antibiotics tetracycline and ampicillin have shown very high MBC values i.e. 3.57 mg/ml and 7.14 mg/ml in
M. luteus,​ while ciprofloxacin has shown very high MBC value i.e.3.57 mg/ml in ​L. acidophilus.​

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 ​Discussion:

​Both herbs and herbal products are known to have antibacterial potential.Herbal
treatments become very popular because it is easily available, cheaper and less toxic than
synthetic drugs. In the present investigation, solvent and aqueous extracts of ​Tinospora
cordifolia a​ nd ​Cassia fistula w
​ ere evaluated for exploration of their antimicrobial activity against
certain Gram negative and Gram positive human pathogenic bacteria. Susceptibility of each plant
extract was tested by serial microdilution method and MIC and MBC values were determined.
Aqueous and solvent extracts of both plant species have shown very high antimicrobial
susceptibility against all seven bacterial strains at a very low concentration. Higher percent
growth inhibition was obtained in presence of plant extracts in comparison to synthetic
antibiotics.. All different extracts have shown very low MIC value, which was found to be lower
(0.0078-0.125 mg/ml) than the MIC values (0.223-0.892 mg/ml) obtained in presence of
standard antibiotic drugs i.e. tetracycline, ampicillin and ciprofloxacin. It proves antimicrobial
susceptibility of plant extracts​. Tinospora cordifolia a​ queous extract has shown lowest MIC
value i.e. 0.00975 mg/ml in ​E. coli​. Similarly ​Cassia fistula ​aqueous extract exhibited a
significant antimicrobial activity against Gram- ve and Gram+ve bacteria. Such inhibitory effects
of in ​C. fistula ​can be attributed to the chemical substances present in the fruits/legumes. It might
be tannins and propyl gallate which are formed in ripening fruits and inhibit the growth of
infectious microorganisms. Further, antimicrobial activity of plant extracts is strengthened by
low MBC values obtained in plant extracts against all seven bacterial strains. However, MBC
values obtained in solvent and aqueous extracts of ​Tinospora cordifolia ​were in range of
0.0292-0.39 mg/ml while these were 0.0156-0.39 mg/ ml in ​Cassia fistula ​in the same extracts.
Further MBC values obtained in plant extracts were significantly much lower than broad
spectrum antibiotics 0.892- 7.17 mg/ml Again it proves antimicrobial potential of plant extracts.

Further, effectiveness of each plant extract was determined by agar disc diffusion method and inhibition
zone diameters obtained in presence and absence of each extract. Based on growth inhibition zone diameter
obtained bacterial strains were divided into three categories i.e. resistant (> 7 mm), intermediate (> 12 mm)
and susceptible (> 18 mm). However, at a very low concentration (20-320 μg/disc) 15- 32 mm inhibition

Page ​32
zone diameters were obtained in chloroform, acetone, metha- nol, petroleum ether and aqueous extracts of
both plant species . These were significantly much larger than the antibiotic drugs i.e. tetracycline, ampicil-
lin and ciprofloxacin which have shown inhibition zone diameters in a range of 6- 18 mm against ​K.
pneumoniae, E. coli, B. cereus​, ​M. luteus, L. acidophilus, S. aureus a​ nd ​S. pneumoniae a​ t the same
concentration. Highest inhibition zone diameter 30 mm was obtained in aqueous extract of ​Cassia fistula
against ​M. luteus at 320 μg/disc. Similar inhibition zone diameter (22 mm) was obtained in methanolic
extract of ​R. coraria​. Results clearly indicate that aqueous and solvent extracts of both plant species were
found to be more effective against both Gram+ve and Gram-ve bacteria.

CONCLUSION

From the present work, it can be concluded that aqueous extracts of above plant species were
found to be more potent which can efficiently reduce the contamination of pathogenic bacteria.
There are two possible explanations for the observed antimicrobial activity. First, the
components are water soluble and bioactive, second these are less soluble in organic solvents. It
is the main reason behind their antimicrobial activity. Here, both plant species have shown nearly
equal antimicrobial effects on both gram positive and gram-negative bacteria in suspension
culture. It is further proved by inhibition zone diameters obtained in filter paper disc diffusion
assays, which have shown better effectiveness of aqueous extracts against Gram-positive
bacteria. It shows the presence of some strong antimicrobial constituents belonging to the
different groups​ ​, which might have very high permeability across the bacterial cell wall due to
ion leakage. There might be another possibility that plant extract may successfully inhibit
microbial respiration and increase the plasma membrane permeability, which results in death of
bacterial cells.

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