phytochemical screening and invitro antibacterial

Kanthimathi and Soranam, 2014/ Antimicrobial activity of Cassia auriculata

International Research Journal of Pharmaceutical and Biosciences (IRJPBS) 2014; 1 (1) : 45-56 45

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:

[email protected]

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 µl 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 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

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

world is rich in biodiversity and most plants were

documented for their medicinal purposes. Herbal

technology is India’s 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

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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

Tanner’s 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

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

at 4oC 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 (Hager’s 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 Benedict’s reagent and heated gently

(Benedict’s 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 Borntrager’s test). Formation of pink

colour indicates the presence of anthranol

glycosides.

Detection of saponins



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

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

tubes were incubated in shaker at 37oC 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

121oC 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 µl) were added into the wells. Well

containing Acetone, Chloroform, Ethanol, Methanol

and Water alone acts as a negative control. The

plates were incubated at 37o 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



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 40µl 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 40µl (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

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



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.

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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.



Table 1: Ethnobotanical data for Cassia auriculata L.

Botanical name

Family

English name

Local name

Part of plant used

Area of

collection

Cassia auriculata

Caesalpiniaceae

Tanners

cassia

Aavarai

Flowers

Azhwarkurichi

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

S.no

Bacteria

Control

10 µl

20 µl

30 µl

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.



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


S.no

Bacteria

Control

10 µl

20 µl

30 µl

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


0

2

4

6

8

10

12

14 D

iam

ete

r o

f zo

ne

s in

mm

Bacterial strains

10µl

20 µl

40 µl



Figure 3 b: Antibacterial effects of methanol extracts of Cassia auriculata against test bacterial


Table 3c: Antibacterial effects of Acetone extracts of Cassia auriculata against test bacterial


S.no

Bacteria

Control

10 µl

20 µl

30 µl

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


0

2

4

6

8

10

12

14

Dia

me

ter

of

zon

e in

mm

Bacterial strains

10µl

20 µl

40 µl



-

Figure 3 c: Antibacterial effects of Acetone extracts of Cassia auriculata against test


Table 3d: Antibacterial effects of water extracts of Cassia auriculata against test bacterial


S.no

Bacteria

Control

10 µl

20 µl

30 µl

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

10µl

20 µl

40 µl 0

2

4

6

8

10

12

14

Dia

me

ter

of

zon

es

in m

m

Bacterial Strains

10µl

20 µl

40 µl



-

Figure 3 d: Antibacterial effects of water extracts of Cassia auriculata against test bacterial


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


S.no

Bacteria

Control

10 µl

20 µl

30 µl

1

Bacillus sp.

-

-

-

-

2

Lactobacillus sp.

-

-

-

-

3

Pseudomonas sp.

-

-

8

9

4

Proteus sp.

-

-

-

-

5

Streptococcus sp.

-

-

-

-

- Sign indicates the absence of Zone

0

2

4

6

8

10

12

Dia

me

ter

of

zon

es

in m

m

Bacterial strains

10µl

20 µl

40 µl



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


10µl

40 µl

0

2

4

6

8

10

12

Dia

me

ter

of

zon

e in

mm

Bacterial strains

10µl

20 µl

40 µl

phytochemical screening and invitro antibacterial

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