INTRODUCTION:
Herbal medicines was found to be known to
man for centuries and they have used some of the traditional medicines to treat common infectious diseases1.
Drug resistance to human pathogenic bacteria and fungus has been reported from all over the world2.
Knowing the antimicrobial properties of plant extract can be of great
significance in therapeutic treatments. In the last few years, a number of
studies have been conducted inorder to prove such efficiency3,4.
The antimicrobial agents of plant origin are not associated with many side
effects and hence it possess an enormous therapeutic potential to cure many
infectious diseases5. Cassia
auriculata belonging to the family Caesalpiniaceae was profoundly used in Ayurvedic
medicine6, known locally as “avaram” or Tanner’s Cassia or Tanner’s
senna . It is a small shrub with smooth brown bark and it is a common plant in
Asia, India and Srilanka. The leaves are useful as anthelmintic, good for
ulcers, leprosy and skin diseases. The flowers have been used in urinary
discharges, diabetes and also for throat infection. The fruit is useful in
thirst and in vomiting. The seed is useful in diabetes, dysentery and chronic
conjunctivitis. The bark is considered as astringent7,8,9.
In the present investigation, an attempt
has been made to enrich the knowledge of antimicrobial activity of ethanolic
extract of the flower and stem parts of C. auriculata Linn.
MATERIALS AND METHODS:
Preparation, collection and
extraction of plant material
The
fresh plant material, stem and flower of Cassia
auriculata was collected from Thiruvanamalai district and Potheri, Chennai
district, Tamilnadu, India. The plant was identified based upon the
organoleptic and macroscopic examination of fresh flower and stem and
authenticated by Prof P. Jayaraman director of Institute of Herbal Botany
,Plant Anatomy Research Center, bearing the reference number PARC/2015/3061.
The collected stems and flowers of Cassia
auriculata is dried thoroughly under shade and powdered mechanically into a
coarse powder. The powdered stem and
flower were kept in airtight container until time of use.
The
flower extract was carried out by continuous hot percolation method using
Soxhlet apparatus10. The solvent used was 95% ethanol. About 50g of
powder was extracted with 400ml of solvent for 72 hours. The extract was
concentrated to dryness under controlled temperature between 40-50şC. The
percentage yield of the Cassia auriculata flower extract (CAFE) was 16.59% w/v.
The
stem extract was carried out by continuous hot percolation method using Soxhlet
apparatus10. The solvent used was 95% ethanol. About 50g of powder
was extracted with 400 ml of solvent. The extract was concentrated to dryness
under controlled temperature between 40-50şC. The percentage yield of the Cassia
auriculata stem extract (CASE) was 12.48% w/v .
Phytochemical Screening of the
extract
The
ethanolic extract of stem and flower were subjected to qualitative test for the
identification of various chemical constituent (alkaloids, flavonoids,
glycosides, proteins, saponins, terpenoids, tannins and phenols)11,12,13..
Antimicrobial Activity
Test Organisms14
Two Gram negative Pseudomonas
aeruginosa, Escherichia coli and two Gram positive Staphylococcus aureus, Bacillus subtilis bacterial pathogens and
one fungal pathogen (Candida albicans)
were used for in vitro antimicrobial activity. These selected pathogenic
strains were obtained from Microbiological Division (Jayagen Biologics
Analytical Laboratory, Chennai).
Preparation of Inoculum
Stock
cultures were maintained at 4°C on Nutrient agar Slant. Active cultures for
experiments were prepared by transferring a loop full of culture from the stock
cultures into the test tubes containing nutrient broth , that were incubated at
24hrs at 37şC. The Assay was performed by agar disc diffusion method.
For Antibacterial Activity
Take
100ml of distilled water in a conical flask and to this add 3.8gm of Mueller Hinton agar (Hi media) and 1 gm of
agar for fast solidification. The flask was tightly plugged with cotton and
wrapped. It was then autoclaved at 15lbs pressure for 20mins. After
sterilization the media was bought to laminar chamber and add antifungal agent
Clotrimazole to avoid bacterial contamination, when the media is half cooled.
Then the media was poured into the petridish for solidification.
Invitro Antibacterial Activity
The antibacterial activity of the flower
and stem extracts of Cassia auriculata
Linn. was determined by disc diffusion methods (CLSI 2000). About 25 mL of molten Mueller Hinton agar was poured
into a sterile Petri plate (Himedia, Mumbai, India). The plates were allowed to
solidify, after which 18 h grown (OD adjusted
to 0.6) 100 µl of above said pathogenic bacteria cultures were
transferred onto plate and made culture lawn by using sterile swab. After five
min setting of the pathogenic bacteria, drug impregnated 5 mm discs were placed
on to the media. The test samples were dissolved in DMSO (5%) and loaded on to
discs with various concentrations such as 50 mg/well,
100 mg/well, 150 mg/well and 200 mg/well.
The 5% DMSO loaded disc served as control. The plates were incubated at 37°C in
a 40 W florescent light source (~ 400 nm) for 24 h. The antibacterial activity
was determined by measuring the diameter of the zone of inhibition around the
well using antibiotic zone scale (Himedia, Mumbai, India) 15
For Antifungal
Activity
Preparation of
Fungal Culture Media
Take
100ml of distilled water in a conical flask and to this add 6.5gm of Sabouraud
Dextroseagar (Hi media) and 1 gm of agar for fast solidification. The flask was
tightly plugged with cotton and wrapped. It was then autoclaved at 15lbs
pressure for 20mins. After sterilization the media was bought to laminar
chamber and add antibacterial agent ampicillin to avoid bacterial contamination
, when the media is half cooled. Then the media was poured into the petri dish
for solidification.
Invitro Antifungal Activity
The antifungal activity was determined by
disc diffusion methods (CLSI 2000).
About 25 mL of molten Sabouraud Dextroseagar was poured into a sterile Petri
plate (Himedia, Mumbai, India). The plates were allowed to solidify, after
which 18 h grown (OD adjusted to 0.6)
100 µl of above said pathogenic fungi cultures were transferred onto plate and
made culture lawn by using sterile swab. After five min setting of the
organism, drug impregnated 5 mm discs were placed on to the media. The test
samples were dissolved in DMSO (5%) and loaded on to dics with various
concentrations such as 50 mg/well, 100 mg/well, 150 mg/well
and 200 mg/well. The 5% DMSO loaded disc
served as control. The plates were incubated at37°C in a 40 W florescent light
source (~ 400 nm) for 24 h. The antifungal activity was determined by measuring
the diameter of the zone of inhibition around the well using antibiotic zone
scale (Himedia, Mumbai, India) 15
MIC Determination
using Microbroth Dilution Assay
Based on the antimicrobial activity of the
stem and flower extract that showed active against Staphylococcus aureus very remarkably, Since both extract were
taken individually for MIC determination using the standard procedure of CLSI
(2012). The microbroth dilution assay was performed to check the MIC for both
extract individually. The S.aureus
has been grown in cation adjusted Mueller Hinton broth and adjusted 5×104cfu/well.
The doubling concentration of the test sample was introduced as 6.25, 12.5, 25,
50, 100, 200, 400 and 800µg/well.
Incubate the inoculated macrodilution tubes or microdilution trays at
35±2°C for 16 to 20 hours in an ambient air incubator within 15 minutes of adding
the inoculum. To prevent drying, seal each tray in a plastic bag, with plastic
tape, or with a tight-fitting plastic cover before incubating. The MIC is the
lowest concentration of antimicrobial agent that completely inhibits growth of
the organism in the microdilution wells as detected by the unaided eye16.
RESULTS:
The
ethanolic extract of stem and flower revealed the presence various chemical
constituents like alkaloids, flavonoids, glycosides, proteins, saponins,
terpenoids, tannins and phenols.
Antimicrobial activity
The
results of antimicrobial activity of ethanolic extract of flower and stem by
well diffusion method was shown in Table 1 and 2. The antimicrobial activity
was measured by zone of inhibition (mm).
The
antibacterial activity of ethanolic extract of flower showed better growth of
inhibition against Staphylococcus aureus (16
mm at 200µg/well) whereas ethanolic extract of stem showed better growth of
inhibition in Escherichia coli (16mm at 200µg/well) when compared to the other
test organisms.
The
antifungal activity of ethanolic extract of flower showed more growth of
inhibition (28mm at 200µg/well) when compared to ethanolic extract of stem
(15mm at 200µg/well).
Table 1: Antimicrobial activity of Flower extract
|
Name of the organisms
|
Zone of the Inhibition (mm)
|
|
50mg/well
|
100mg/well
|
150mg/well
|
200mg/well
|
|
Staphylococcus aureus
|
9
|
11
|
14
|
16
|
|
Bacillus subtilis
|
8
|
10
|
12
|
14
|
|
Pseudomonas aeruginosa
|
-
|
8
|
11
|
14
|
|
Escherichia coli
|
7
|
9
|
12
|
15
|
|
Candida albicans
|
13
|
17
|
21
|
28
|
Table 2: Antimicrobial activity of Stem extract
|
Name of the organisms
|
Zone of the Inhibition (mm)
|
|
50mg/well
|
100mg/well
|
150mg/well
|
200mg/well
|
|
Staphylococcus aureus
|
7
|
9
|
11
|
14
|
|
Bacillus subtilis
|
-
|
-
|
8
|
12
|
|
Pseudomonas aeruginosa
|
7
|
8
|
9
|
11
|
|
Escherichia coli
|
7
|
9
|
12
|
16
|
|
Candida albicans
|
7
|
9
|
12
|
15
|
MIC assay
The
MIC determination of the both stem and flower showed minimum inhibitory
concentration level of increased concentration. Ethanolic extract of flower
showed MIC at 200 µg killed entire S. aureus inoculated well after 20 h of
incubation whereas ethanolic extract
of stem showed MIC at 400 µg that killed the entire 5×104
CFU/well at 20 h of incubation.
DISCUSSION:
The medicinal property and pharmacological
action of Cassia auriculata is well
used in the Indian traditional medicine. These plants are known to contain
various active principle of therapeutic value and to possess biological
activity against a number of diseases.
Cassia auriculata
(linn)
belonging to the family Caesalpiniacea, is commonly known as “Avaram” in Tamil,
“Tangedu” in Telugu, “tanner’s cassia” in English and “avara” in Malayalam. The
flower and stem parts was taken for the present study and ethanolic extract
were prepared. The work was planned to investigate the antimicrobial activity of flower and stem extract using various
organisms such as two Gram negative organisms (Pseudomonas aeruginosa and
Escherichia coli) and two
Gram positive organisms, (Staphylococcus
aureus and Bacillus subtilis) and
one fungal pathogen (Candida albicans).
The
ethanolic extract of flower and stem were prepared and their yield were noted.
The preliminary phytochemical test were performed on the ethanolic extract of
stem and flower and showed the presence of various chemical constituents like
alkaloids, flavonoids, glycosides, proteins, saponins, terpenoids, tannins and
phenols.
The
zone of inhibition were measured after 24 hours of incubation and the result
were tabulated (Table-1and2). The ethanolic flower extract showed more activity
in Staphylococcus aureus (zone of
inhibition-16 mm at 200µg/well and ethanolic extract of stem showed more
activity in Escherichia coli (zone of
inhibition-16mm at 200µg/well) when compared to other test organisms. The
antifungal activity of ethanolic extract of flower showed more inhibition of
growth (28mm at 200µg/well) when compared to ethanolic extract of stem (15mm at
200µg/well).
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