Kanthimathi and Soranam, 2014/ Antimicrobial activity of Cassia auriculata

ORIGINAL ARTICLE

International Research Journal of Pharmaceutical and Biosciences

Pri -ISSN: 2394 5826

http://www.irjpbs.com e-ISSN: 2394 - 5834

Phytochemical screening and Invitro antibacterial Potential

of Cassia auriculata Linn. Flowers Against Pathogenic Bacteria
M. Kanthimathi and R. Soranam*

Sri Paramakalyani Centre for Environmental Sciences, Manonmaniam sundaranar University, Alwarkurichi-
627412, Tamilnadu, India.

Article info Abstract

Article history:
Received 12September 2014
Accepted 18 October 2014
*Corresponding authors:
E-mail address:
kanthibt@gmail.com
Copyright 2014 irjpbs
The aim of present study is to investigate the screening of phytochemicals and to
determine the antibacterial potential of Cassia auriculata flowers against five
human bacterial pathogens namely Bacillus sp., Lactobacillus sp., Pseudomonas
sp., Proteus sp., and Streptococcus sp. using five different solvents namely,
acetone, chloroform, ethanol, methanol and water. The phytochemical analysis
gave the positive result for Alkaloids, Saponin, Terpenoids, Phenols, Tannins,
Flavonoids, Carbohydrates, Proteins and Amino acids. The maximum antibacterial
activities were assessed with agar well diffusion method. 10, 20, 40 l volumes of
different plant extracts were used. The antibacterial activity decreased in the
order of Ethanol > Methanol > Acetone > Water > Chloroform. Out of the five
extracts used methanol and ethanol were found to be highly active against
Bacillus sp., Lactobacillus sp., and Streptococcus sp. Moderate antibacterial
potential was seen in acetone and aqueous extracts and no bacterial activity was
recorded with chloroform extracts except for Proteus sp.
Key words: Cassia auriculata, Phytochemical analysis, Agar well diffusion method,
Zone of Inhibition, Antibacterial activity

INTRODUCTION antimicrobials [2]. India, the botanical garden of the

Medicinal plants constitute the basis of primary
health care for majority of population in world. The
plants are vital sources for development of potent
antimicrobial drugs, they are continued to play a
dominant role in maintenance of human health since
ancient days [1].The World Health Organization has
estimated that 80% of people in Asia and Africa rely
on herbal medicines and 25 to 50 % of
pharmaceuticals bestowed around the world are of
plant origin and few among them have been used as
world is rich in biodiversity and most plants were
documented for their medicinal purposes. Herbal
technology is Indias one of the biggest revenue
sources [3]. The potential of plants as a source of
drugs is largely uncharted. The medicinal properties
of the plants are mainly due to the antioxidant,
antimicrobial and antipyretic effect of the
phytochemicals present in them [4]. The medicinal
properties of a plant is mainly due to the presence of

International Research Journal of Pharmaceutical and Biosciences (IRJPBS) 2014; 1 (1) : 45-56 45
 Kanthimathi and Soranam, 2014/ Antimicrobial activity of Cassia auriculata
phytochemicals like alkaloids, glycosides, volatile oils,
tannins, saponins, etc. [5] The plants are rich source
of antimicrobial agents. Thus it is significant to carry
out a study on medicinal plants for their antimicrobial
potential. Studies revealed that traditional medicines
provides essential oils and other plant extracts that
provoke the interest as sources of natural products
for its potentiality as an alternate remedy for various
infectious diseases [6]. A detailed investigation about
the antimicrobial properties of the plants that are
used local traditions can leads to the development of
invaluable plant drugs for dreadful diseases.
The purpose of the present study was to determine
the presence of phytoconstituens and to investigate
the antibacterial properties of flowers of Cassia
auriculata. Cassia auriculata commonly known as
Tanners Cassia and Aavaarai in tamil is a weed
widely distributed in dry regions of Madhya Pradesh,
Tamil Nadu and Rajasthan. It belongs to the family
Caesalpiniaceae. The plants produces flowers in
bunches. The flowers are irregular, bisexual, The
petals and sepals are numbered five and are free,
crisped along the margin, bright yellow veined with
orange and large (nearly 5cm across), the pedicles
glabrous and 2.5cm long. The anthers are around
ten in numbers and are separate, with the three
upper stamens barren; the ovary is superior,
unilocular with marginal ovules [7].
The present communication attempts to study the
antimicrobial activity of separate extraction using
acetone, chloroform, ethanol, methanol and
aqueous extracts from flowers of Cassia auriculata
which is investigated against a few bacterial
pathogens using agar well diffusion method and to
report the phytoconstituents present in different
extracts of flowers of Cassia auriculata.
MATERIALS AND METHODS
Plant material selection
The flowers of Cassia auriculata (Fig 1) used for the
present study were collected in the campus of Sri
Paramakalyanic Centre for Environmental Sciences,
Manonmaniam Sundaranar University,
Azhwarkurichi, Tamilnadu, India and were identified
based on its physical characteristics. The
ethnobotanical data of Cassia auriculata were given
in table 1. The flowers were ensured that they were
healthy and uninfected and they were thoroughly
International Research Journal of Pharmaceutical and Biosciences (IRJPBS) 2014; 1 (1) : 45-56
washed with tap water and rinsed with sterile
distilled water and air dried in room temperature.
Then the flowers were crushed to small pieces using
pestle and mortar.
Preparation of extracts
Twenty five grams of the powdered flowers of
Cassia auriculata were extracted successively with
50 ml Acetone, chloroform, Ethanol, Methanol and
Water using soxhlet extractor. The obtained extracts
were concentrated in rotary vaccum evaporator and
the resulting pasty form of the extracts were stored
o
at 4 C in air tight container for further use.
Preliminary phytochemical analysis
The Acetone, chloroform, Ethanol, Methanol and
Aqueous extracts of flowers of Cassia auriculata
were analyzed by the following procedures [8, 9] for
the presence of alkaloids, glycosides, saponin,
terpenoids, triterpenes, phenols, tannins,
phlobatannins, flavonoids, carbohydrates, proteins
and amino acids.
Detection of alkaloids
The extracts were hydrolyzed individually in dilute
Hydrochloric acid and treated with saturated picric
acid solution (Hagers test). Presence of alkaloids
was confirmed by the presence of yellow coloured
precipitate.
Detection of carbohydrates
The extracts were dissolved in distilled water and
treated with Benedicts reagent and heated gently
(Benedicts test). An orange red precipitate indicates
the presence of reducing sugars.
Detection of glycosides
The extracts were hydrolyzed with dilute
Hydrochloric acid and then treated with Ferric
chloride solution and kept in boiling water for five
minutes. The mixture was cooled and equal volume
of benzene was added. The benzene layer was
separated and treated with ammonia solution
(Modified Borntragers test). Formation of pink
colour indicates the presence of anthranol
glycosides.
Detection of saponins
46
 Kanthimathi and Soranam, 2014/ Antimicrobial activity of Cassia auriculata
The extracts were diluted with distilled water to 10
ml and this was shaken for about 15 minutes.
Formation of foam that persists for few minutes
confirms the presence of saponins.
Detection of phytosterols
The extracts were treated with chloroform and then
filtered. A few drops of concentrated sulphuric acid
were added, Shaken and kept. The golden yellow
colour formation indicates the presence of
triterpenes.
Detection of steroids
The extracts were treated with 5 ml of chloroform
and equal volume of concentrated sulphuric acid
were added along the sides of the test tubes,
formation of yellowish green colour indicates the
presence of steroids.
Detection of terpenoids
The extracts were treated with chloroform, filtered
and treated with few drops of concentrated
Sulphuric acid, shaken and allowed to stand. Change
of reddish brown color revealed the presence of
terpenoids.
Detection of phenols
The extracts were treated with three to four drops
of Ferric chloride solution. The bluish black colour
indicates the presence of phenols.
Detection of tannin
The extracts were treated with equal volume of 1%
Ferric chloride solution, Brownish green colour
formation indicates the presence of tannin.
Detection of flavonoids
To the extracts few drops of 20% Sodium Hydroxide
solution were added, which results in yellow colour
formation that disappears while adding dilute acid
indicates the presence of flavonoids.
Detection of phlobatannins
The extracts were treated with equal volume of 1%
Hydrochloric acid and boiled. Red precipitate
indicates the presence of phlobatannins.
Detection of proteins
International Research Journal of Pharmaceutical and Biosciences (IRJPBS) 2014; 1 (1) : 45-56
The extracts were treated with few drops of
concentrated nitric acid. Yellow colour indicates the
presence of proteins.
Detection of aminoacids
The extracts were treated with 0.25% Ninhydrin
reagent and boiled for few minutes. Formation of
blue colour indicates the presence of aminoacids.
Antibacterial activity
Test microorganisms
Clinical isolates of Bacillus sp., Lactobacillus sp.,
Pseudomonas sp., Proteus sp., and Streptococcus sp.
were obtained as stock culture from the Department
of Microbiology, Sri Paramakalyani College,
Azhwarkurichi, Tamil Nadu.
Preparation of inoculums
Active cultures for experiments were prepared by
transferring a loopfull of cells from the stock
cultures to test tubes of Nutrient broth and the
o
tubes were incubated in shaker at 37 C for 24 hrs
that were used as the inoculums.
Antibacterial activity
Antibacterial activity of the extracts were
determined by agar well diffusion method [10]. The
Muller Hinton agar was sterilized in autoclave at
o
121 C at 15 lbs and it was poured in clean, sterile
petri dishes in uniform thickness under aseptic
condition. After solidification, the plates were
inoculated with active bacterial cultures by spread
plate method. The excess of inoculum were
removed with micropipettes aseptically. The wells
were made with sterile cork borer of 6mm diameter
and the cut agar discs were removed with sterile
foreceps. Acetone, Chloroform, Ethanol, Methanol
and Water extracts with different volume (10, 20
and 40 l) were added into the wells. Well
containing Acetone, Chloroform, Ethanol, Methanol
and Water alone acts as a negative control. The
o
plates were incubated at 37 C for 24 hours. The
antibacterial activity was assessed by measuring the
diameter zones of inhibition (ZOI) in millimeter.
RESULTS
The ethanobotanical data in Table 1 includes the
scientific, family, English and local name of C.
auriculata, whose extracts were screened for
47
 Kanthimathi and Soranam, 2014/ Antimicrobial activity of Cassia auriculata
phytochemical constituents and for antibacterial
assay. The phytochemical characteristics of the five
different extracts of C. auriculata flowers
investigated are summarized in Table 2. This Primary
screening test revealed the presence of medicinally
active constituents in the five extracts of C.
auriculata flower studied. From Table 2, the study
indicated that Proteins were present in all extracts
while, glycosides, triterpenes, and phlobatannins
were absent in all the five extracts. carbohydrates,
terpenoids and steroids were absent only in
chloroform extract. Saponin, was absent in both
chloroform and methanol extracts. Phenols and
flavonoids were absent in both chloroform and
aqueous extracts. Amino acids were absent in
acetone and ethanol extracts. And alkaloids and
tannins were present only in aqueous extract.
The qualitative antibacterial assay revealed that out
of the five different extracts, ethanol and methanol
extracts of C. auriculata flowers posses antibacterial
activity against Bacillus sp., Lactobacillus sp.,
Streptococcus sp. Among the various solvents
tested, the ethanol and methanol showed highest
inhibitory activity when compared to other solvents.
Next to them, acetone extract showed good
inhibitory activity followed by aqueous extract and
the inhibitory activity of chloroform extract was
relatively low when compared to the other tested
solvent extracts. The acetone extract was found to
be active against Bacillus sp., Proteus sp., and
Streptococcus sp. And the chloroform extract was
little active against the Pseudomonas sp. only. The
results of these bacterial bioassays were shown in
Fig 2. The antibacterial activity of different extracts
of Cassia auriculata determined by agar well
diffusion method was shown in Table 3 a-e. Highest
zone of inhibition (14 mm) was observed against
Bacillus sp., and Lactobacillus sp. at 40l of acetone
and ethanol extracts respectively (Table 3a, 3b and
Fig 3a, 3b). The Proteus sp. was sensitive against
acetone extract only with moderate ZOI of 11.5 mm
diameter at volume of 40l (Table 3c and Fig 3c) for
other extracts it was found to be resistant even at
higher concentrations also. The aqueous extract has
very moderate antibacterial effect against Bacillus
sp., and Streptococcus sp. (Table 3d and Fig 3d). The
chloroform extract was not active against all other
bacterial strains except for Pseudomonas sp. and
lowest ZOI of 9 mm diameter was only observed
International Research Journal of Pharmaceutical and Biosciences (IRJPBS) 2014; 1 (1) : 45-56
against Pseudomonas sp. (Table 3e and Fig 3e). The
variations in the activities of different extracts may
be due to insolubility of active phytochemicals or
the presence of any inhibitors to the antimicrobial
compounds.
DISCUSSION
In present study the flower extracts of Cassia
auriculata were undertaken to do preliminary
screening of phytochemical constituents and to
evaluate their probable role as an antibacterial
agent due to resistance developed against
widespread of marketed antibiotic formulations. The
preliminary phytochemical investigations were
qualitative and it was observed that alkaloids,
saponin, terpenoids, phenols, tannins, flavonoids,
carbohydrates, proteins and amino acids were
present in the extracts. The ethanol and methanol
extracts of Cassia auriculata showed more inhibitory
effect than the other extracts. This tends to show
that the active ingredients of the plant parts are
better extracted with ethanol and methanol than
acetone, chloroform and aqueous extracts. The C.
auriculata flowers are known to contain various
active principle of therapeutic value and possess
biological activity against various diseases [11]. The
antimicrobial properties of C.auriculata extract may
be due to the presence of phenolic constituents
[12]. The methanol extracts contains, saponins,
flavonoids, alkaloids, tannins and phenolic
compounds the phytochemicals that are responsible
for their antibacterial properties [13]. Cassia
auriculata had the potent inhibitory action against
fungal strains than bacterial strainst tested and it
showed strong antifungal properties when
compared to Fluconazole, a standard antifungal drug
[14]. The ethanol and methanol extracts of dry and
fresh flowers, aqueous extract of dry flower and
acetone extract of flower was found to have
inhibitory activities against E. fecalis, S. aureus, B.
subtilis, E.coli, P. mirabilis, S. typhi, V. cholera and S.
dysenteriae. The presence of phytochemicals like
terpenoids, tannins, flavonoids, saponin, cardiac
glycosides and are responsible for its antibacterial
activity [15].
CONCLUSION
In this study the results suggested that different
extracts under study showed different antimicrobial
48
 Kanthimathi and Soranam, 2014/ Antimicrobial activity of Cassia auriculata
activity. The antibacterial action of various extracts
of flowers of C. auriculata may indicate their
potential as antibacterial herbal remedies. The
preliminary results of our investigation indicate that
C. auriculata flowers have high potential of
antimicrobial activity. Further work is needed to
carryout pharmacological investigations to support
the use of this herbal plant.
REFERENCES
1. E. O. Farombi, African indigenous plants with
chemotherapeutic potentials and biotechnological
approach to the production of bioactive
prophylactic agents, African Journal of
Biotechnology. 2 (2003) 662-671.
2. P. Bharathi, Alex Thomas, Ansa Thomas, S.
Krishnan and T. K. Ravi, Anti bacterial activity of
leaf extracts of Calotropis gigantea Linn. against
certain gram negative and gram positive bacteria,
Int. J. Chem. Sci. 9(2011) 919-923.
3. A.Sharma, C. Shanker, L. Tyagi, M. Singh and C.V.
Rao, Herbal medicine for market potential in India:
An Overview, Academic Journal of Plant Sciences.
1(2008) 26-36.
4. A.A. Adesokan, M.T. Yakubu, B.V. Owoyele, M.A.
Akanji, A. Soladoye and O.K. Lawal, Effect of
administration of aqueous and ethanolic extracts
of Enantia chlorantha stem bark on brewers yeast-
induced pyresis in rats, Afri. J. Biochem. Res.
2(2008) 165-169.
5. Satheesh Kumar Bhandary , N. Suchetha Kumari,
Vadisha S. Bhat , K.P Sharmila and Mahesh Prasad
Bekal, Preliminary Phytochemical Screening of
Various Extracts of Punica granatum Peel, Whole
Fruit and Seeds, NUJHS 2(2012) 34-38.
6. C.K. Hindumathy, Invitro Study of Antibacterial
Activity of Cymbopogon Citratus, World Academy
of Science. Engineering and Technology 50 (2011)
189-193.
7. V.joy, M. Paul John Peter, J. Yesu Raj, and Ramesh,
Medicinal values of avaram (Cassia auriculata
International Research Journal of Pharmaceutical and Biosciences (IRJPBS) 2014; 1 (1) : 45-56
Linn.): a review, International Journal of Current
Pharmaceutical Research. 4(2012) 1-3.Prashant
Tiwar, Bimlesh Kumar, Mandeep Kaur, Gurpreet
Kaur and Harleen Kaur, Phytochemical screening
and Extraction: A Review, Internatioanle
Pharmaceutica Sciencia, 1(2011) 98-106.
8. O. Victor Njoku and Chidi Obi, Phytochemical
constituents of some selected medicinal plants,
African Journal of Pure and Applied Chemistry, 3
(2009) 228-233.
9. M. Cheesbrough, Culture media In: Medical
Laboratory Manual for tropical countries, Tropical
Health Technology and Butterworth- Heineman.
Cambridge. (1994) 60-69.
10. M. Ayyanar, and Ignacimuthu, Pharmacologica
Actions of Cassia quadrangular are wall: A short
Review, J. Pharmacol. Toxicol. 3(2008): 213-221.
11. H. Mohamed Sham Shihabudeen, D. Hansi Priscilla,
S.S. Kavitha and Thirumurugan, Antimicrobial
activity and phytochemical analysis of selected
Indian folk medical plants, International Journal of
Pharma science and research (IJPSR). 1(2010): 430-
434.
12. Gaurav M. Doshi, S. Supriya, Shidhaye, V. Gayatri,
Aggarwal , Preeja P. Pillai, Abhijeet B. Bhalerao,
Sandhya K. Desai, Antibacterial Potential Of Cassia
auriculata Flowers, Journal of Microbiology and
Biotechnology Research. 1(2011) 15-19.
13. P.Muthukumaran, M.Elayarani,
P.Shanmuganathan and A.Cholarajan,
Antimicrobial Activities of Cassia auriculata L and
Morinda tinctoria Roxb, International Journal of
Research in Pure and Applied Microbiology.
1(2011) 9-12.
14. P. Sujith and P.K. Senthilkumar, Bactericidal
Activity of Extracts of Different Flowering Stages of
Cassia auriculata and Screening of its Amino Acids,
International Journal of Microbiological Research.
3(2012) 144-148.
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 Kanthimathi and Soranam, 2014/ Antimicrobial activity of Cassia auriculata

Figure 1: Cassia auriculata Linn. Flowers

a Bacillus sp., b- Lactobacillus sp., c- Psedomonas sp., d- Proteus sp.,

e- Streptococcus sp., f- Control

Figure 2: Preliminary screening for antibacterial activity of M- Methanol extract, E-

Ethanol extract, A- Acetone extract, C- Chloroform extract and W- Aqueous extract of
Cassia auriculata Linn. Flowers.

International Research Journal of Pharmaceutical and Biosciences (IRJPBS) 2014; 1 (1) : 45-56 49
 Kanthimathi and Soranam, 2014/ Antimicrobial activity of Cassia auriculata

Table 1: Ethnobotanical data for Cassia auriculata L.

Botanical Family English Local Part of plant Area of

name name name used collection

Cassia auriculata Caesalpiniaceae Tanners Aavarai Flowers Azhwarkurichi

cassia

Table 2: Qualitative phytochemical evaluation

Phytochemicals Acetone Chloroform Ethanol Methanol Aqueous

Alkaloids - - - - +
Carbohydrate + - + + +
Glycosides - - - - -
Saponin + - + - +
Triterpenes - - - - -
Terpenoids + - + + +
Phenols + - + + -
Tannins - - - - +
Phlobatannins - - - - -
Flavonoids + - + + -
Steroids + - + + +
Proteins + + + + +
Aminoacids - + - + +

+ = Present, - =Absent
Table 3a: Antibacterial effects of Ethanol extracts of Cassia auriculata against test
bacterial pathogens in different concentration

10 l 20 l 30 l
S.no Bacteria Control

1 Bacillus sp. - 10 12 13

2 Lactobacillus sp. - 12 12 14

3 Pseudomonas sp. - - - -

4 Proteus sp. - - - -

5 Streptococcus sp. - 11 11.5 12

- Sign indicates the absence of Zone.

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 Kanthimathi and Soranam, 2014/ Antimicrobial activity of Cassia auriculata

14

12
Diameter of zones in mm

10

8
6
4 10l

2 20 l

0 40 l

Bacterial strains

Figure 3 a: Antibacterial effects of Ethanol extracts of Cassia auriculata against test bacterial
pathogens in different concentration

Table 3b: Antibacterial effects of Methanol extracts of Cassia auriculata against test bacterial
pathogens in different concentration

10 l 20 l 30 l
S.no Bacteria Control

1 Bacillus sp. - 9 9.5 11

2 Lactobacillus sp. - 9 11 13

3 Pseudomonas sp. - - - -

4 Proteus sp. - - - -

5 Streptococcus sp. - 10.5 12 12

- Sign indicates the absence of Zone.

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 Kanthimathi and Soranam, 2014/ Antimicrobial activity of Cassia auriculata

14

Diameter of zone in mm
12
10
8
6
4
2 10l
0
20 l
40 l

Bacterial strains

Figure 3 b: Antibacterial effects of methanol extracts of Cassia auriculata against test bacterial
pathogens in different concentration

Table 3c: Antibacterial effects of Acetone extracts of Cassia auriculata against test bacterial
pathogens in different concentration

10 l 20 l 30 l
S.no Bacteria Control

1 Bacillus sp. - 13.5 14 14

2 Lactobacillus sp. - - - -

3 Pseudomonas sp. - - - -

4 Proteus sp. - 10 11 11.5

5 Streptococcus sp. - - 12 12
- Sign indicates the absence of Zone.

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 Kanthimathi and Soranam, 2014/ Antimicrobial activity of Cassia auriculata

14

Diameter of zones in mm
12
10
8
6
4 10l
2 20 l
40 l
0 40 l
20 l
10l

Bacterial Strains

-
Figure 3 c: Antibacterial effects of Acetone extracts of Cassia auriculata against test
bacterial pathogens in different concentration

Table 3d: Antibacterial effects of water extracts of Cassia auriculata against test bacterial
pathogens in different concentration

10 l 20 l 30 l
S.no Bacteria Control

1 Bacillus sp. - 7.5 9 11

2 Lactobacillus sp. - - - -

3 Pseudomonas sp. - - - -

4 Proteus sp. - - - -

5 Streptococcus sp. - - 8 9.5

- Sign indicates the absence of Zone

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 Kanthimathi and Soranam, 2014/ Antimicrobial activity of Cassia auriculata

12

10
Diameter of zones in mm

4 10l

2 20 l
40 l
0

Bacterial strains

Figure 3 d: Antibacterial effects of water extracts of Cassia auriculata against test bacterial
pathogens in different concentration

Table 3e: Antibacterial effects of Chloroform extracts of Cassia auriculata against test
bacterial pathogens in different concentration

10 l 20 l 30 l
S.no Bacteria Control

1 Bacillus sp. - - - -

2 Lactobacillus sp. - - - -

3 Pseudomonas sp. - - 8 9

4 Proteus sp. - - - -

5 Streptococcus sp. - - - -

- Sign indicates the absence of Zone

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 Kanthimathi and Soranam, 2014/ Antimicrobial activity of Cassia auriculata

12

10
Diameter of zone in mm

6 10l

4 20 l

40 l 40 l
2
10l
0

Bacterial strains

Figure 3 e: Antibacterial effects of Chloroform extracts of Cassia auriculata against test

bacterial pathogens in different concentration

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