323 Sowjanya Pulipati. et al. / International Journal of Biological & Pharmaceutical Research. 2014; 5(4): 323-326.

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QUANTITATIVE DETERMINATION OF TANNIN CONTENT AND

EVALUATION OF ANTIBACTERIAL ACTIVITY OF CROSSANDRA

INFUNDIBULIFORMIS (L) NEES AGAINST UTI PATHOGENS

Sowjanya Pulipati*, Vamsi Anil Krishna Chandu, Reshma Begum, Srinivasa Babu P

Vignan Pharmacy College, Vadlamudi- 522 213, Guntur (Dt), Andhra Pradesh, India.

ABSTRACT

The present study is designed to determine the tannin content and evaluation of antibacterial activity of Crossandra

infundibuliformis (Acanthaceae) flower extracts against pathogens associated with urinary tract infections. The Urinary Tract

Infections represent one of the most common diseases occurring from the neonate to the geriatric age groups encounters in

medical practice today. The chloroform, ethyl acetate, acetone and aqueous extracts for the plant material were prepared. The

extracts contained appreciable levels of tannin content (2.7 to 8.6 mg of GAE/gm extract). Among all the extracts acetone

extract possess higher concentration of tannins. Leading etiological agents of UTI’s include Escherichia coli, Enterococcus

faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus vulgaris. The susceptibility patterns of these UTI

pathogens against extracts were tested. The antibacterial activity was prominent against Escherichia coli (16.20±0.25mm) by

acetone extract. The results indicated that natural tannin from flowers of Crossandra infundibuliformis has a notable

antibacterial activity against tested microorganisms. The results are compared with commercial antibiotic nitrofurantoin.

Key Words: Crossandra infundibuliformis, Tannin content, Antibacterial, UTI pathogens.

INTRODUCTION

A urinary tract infection is an infection that

affects part of the urinary tract. UTI is more common in

women than men. In women it occurs in all age groups but

the incidence and prevalence increase with age. It is the

second most common bacterial infection occurs during

pregnancy (Sampson JE, Gravett MG, 1999). Lower UTI is

also referred to as a bladder infection. The symptoms are

burning with urination and urge to urinate frequently

(Nicolle LE, 2008). Upper UTI is also referred as

pyelonephritis and the symptoms are flank pain, fever,

nausea or vomiting in addition to lower UTI symptoms

(Lane DR, Takhar SS, 2011). Around 80-85% of urinary

tract infections are caused by E. Coli and 5-10% by

Staphylococcus saprophyticus (Nicolle LE, 2008). Rarely

Corresponding Author

Sowjanya Pulipati

Email: sowjypulipati@gmail.com

the UTI infections are due to viral or fungal infections

(Amdekar S, 2011). The other bacteria that cause UTI

infections are Klebsiella, Proteus, Pseudomonas and

Enterobacter.

For centuries plants have been used as drugs and

remedies for various diseases. They are the best source to

obtain a variety of drugs. Plants accumulate various

secondary metabolites including alkaloids, glycosides,

polyphenols. Tannins are water soluble polyphenols;

widely distributed in almost all plants. They possess

astringent property. They have been classified into two

groups: hydrolysable and condensed tannins (Romani et

al., 2006; Buzzini et al., 2008). Hydrolysable tannins are

based on gallic acid, usually as multiple esters with D-

glucose, while condensed tannins (often called

proanthocyanidins) are derived from flavonoid monomers.

Human physiological activities like stimulation of

phagocytic cells, host mediated tumour activity and anti-

IJBPR

324 Sowjanya Pulipati. et al. / International Journal of Biological & Pharmaceutical Research. 2014; 5(4): 323-326.

infective activities have been assigned to tannins. One of

their mechanisms is to complex with proteins through non-

specific forces (Haslam E, 1996). The tannin containing

remedies are used as anthelmintics, antioxidants and

antimicrobial agents. To promote the proper use and to

determine the potential of plants as sources for new drugs,

it is essential to study the medicinal plants to understand

their properties, safety and efficacy.

Crossandhra infundibuliformis (Acanthaceae) is

an important plant in horticulture (Fig:1). It is abundantly

present in tropical areas such as South India and Sri Lanka.

The leaf extract of Crossandhra infundibuliformis shows

aphrodisiac activity (Kumar SA et al., 2010).The ethanolic

leaf extract of C.infundibuliformis showed anti-

inflammatory and analgesic properties (Jayaveera KN et

al., 2012). The leaf extracts possess antibacterial,

antioxidant activities (N. Sharmila et al., 2011). The

ethanol extract of C.infundibuliformis leaves exhibited

good antioxidant activity. The aqueous and methanol leaf

extracts of C. infundibuliformis possess antimicrobial

activity tested against some common bacterial and fungal

pathogens (Elamanthi R et al., 2011). The petroleum ether

leaf extract of Crossandra infundibuliformis L. possess

wound healing activity in excision wound model

(Sumalatha K, 2012). Due to its medicinal value, this plant

is used to treat various ailments.

MATERIALS AND METHODS

Plant Material

The fresh flowers of Crossandra

infundibuliformis were collected and authenticated from

Department of Botany, Acharya Nagarjuna University,

Guntur, Andhra Pradesh. The healthy flowers were shade

dried and powdered using electric blender to get a coarse

powder.

Extraction Process

The powdered material was extracted with

solvents like chloroform, ethyl acetate, acetone by cold

maceration process. The extracts were prepared by taking

15g of dried flower powder in separate containers and to

this 150mL of each solvent was added and kept in a shaker

for 24 h. The aqueous extract was prepared by boiling 15g

of powder with 150ml of distilled water at mild

temperature for 20 minutes. The extracts were collected by

filtration through 5 layers of muslin cloth. The extraction

process was repeated twice. The collected filtrates were

pooled, concentrated and dried at mild temperature

(Maneemegalai S and Naveen T, 2010).

Phytochemical Screening

The phytochemical screening for the extracts was

carried out by standard protocols (Evans WC, Trease and

Evans, 2005, Kokate CK et al., 2005). Alkaloids (Mayer’s

test), glycosides (Legal’s test), saponins (froth formation

test), carbohydrates (Molisch’s test), proteins

(Xanthoproteic test), aminoacids (Ninhydrin test),

Flavonoids (Lead acetate test), steroids (Salkowski test),

tannins (Ferric chloride test) were analyzed.

Determination of Total Phenolic Content

The total phenolic content was determined using

Folin Ciocalteau reagent. A standard calibration curve was

prepared and the absorbance against concentration of

tannins at 725nm was estimated spectrophotometrically.

Gallic acid was used as a standard and the total phenolic

content was expressed as µg/ml gallic acid equivalents

(GAE). Concentration of 1mg/ml of plant extract was

prepared in methanol and 0.5ml of each sample were

introduced into test tubes and mixed with 0.5ml of a 1N

dilute Folin-Ciocalteau reagent and 2.5ml of 20% sodium

carbonate. The tubes were covered with parafilm and

allowed to stand for 40 minutes at room temperature and

absorbance was read at 725nm spectrophotometrically

(Koleckar V et al., 2008).

Determination of Tannin Content

Tannin content was determined using insoluble

polyvinyl-polypyrrolidone (PVPP), which binds tannins.

Briefly 1ml of extract (1mg/ml) in which the total

phenolics was determined, was mixed with 100mg of

PVPP, vortexed, kept for 15min at 40C and then

centrifuged for 10 min at 3000 rpm. In the clear

supernatant non-tannin phenolics were determined the

same way as that of total phenolics. Tannin content was

calculated as a difference between total and non-tannin

phenolic content.

Agar Well Diffusion Method

The present study was designed to determine the

susceptibility pattern of chloroform, ethyl acetate, acetone

and aqueous extracts utilizing UTI pathogens like

Escherichia coli, Pseudomonas aeruginosa, Klebsiella

pneumoniae, Enterococcus faecalis, Proteus vulgaris by

agar well diffusion method. To check the susceptibility

pattern of extracts against bacteria the Muller Hinton agar

media was inoculated with specific organisms. The wells

of 6 mm diameter were made equidistantly in the agar plate

with sterile borer. Each well is filled with 10µl

(concentration 50mg/ml) of the extract. The plates were

incubated in upright position for 24 h and zones of

inhibition were measured. The activity was compared with

nitrofurantoin (300µg/ml)

RESULTS & DISCUSSION

Phytochemical Screening

The results of preliminary phytochemical

screening revealed the presence of various

phytoconstituents (Table 1). The carbohydrates and cardiac

glycosides were present in ethyl acetate and acetone

extracts. The alkaloids were present in ethyl acetate,

325 Sowjanya Pulipati. et al. / International Journal of Biological & Pharmaceutical Research. 2014; 5(4): 323-326.

acetone and aqueous extracts. The presence of tannins and

flavonoids were observed in chloroform, ethyl acetate,

acetone and aqueous extracts.

Tannins

The presence of tannins in the plants exhibited

various biological activities like antibacterial, antifungal,

anthelmintic. Acetone extract possess highest tannin

content 8.6 mg of GAE/gm. Chloroform and aqueous

extracts possess moderate amount of tannin content 6 and

5.4 mg of GAE/gm whereas ethyl acetate extract possess

less amount of tannin content 2.7 mg of GAE/gm. The

results of total phenolic and tannin content were

represented in table:2. The total phenolic content and non

phenolic content were estimated through the standard

calibration curve of gallic acid (Fig 2).

Antibacterial Activity

The present study shows the evaluation of in vitro

antimicrobial activity of Crossandra infundibuliformis

against Gram-positive, Gram-negative UTI pathogens

using agar well diffusion method. The results of

antibacterial activity were given in table-3, which clearly

show that all the extracts have shown good antibacterial

activity equivalent to that of the standard against entire

tested organisms. Antibacterial activity was confirmed by

the presence of zone of inhibition.

Results of antibacterial assay revealed that

acetone extract of plant exhibited prominent antibacterial

activity against tested UTI isolates compared to

chloroform, ethyl acetate and aqueous extracts. The

susceptibility of pathogens towards extracts varied to

solvent used for extraction. The acetone extract exhibited

maximum antibacterial activity especially against E.coli,

moderate activity against P. aeruginosa and minimum

activity against K. pneumonia, E.faecalis, P. vulgaris. The

chloroform extract exhibited highest antibacterial activity

against E.faecalis and lowest activity against E.coli. The

maximum activity of ethyl acetate extract was observed

against E.faecalis and minimum activity against

P.vulgaris. The aqueous extract showed highest activity

against E.coli and least antibacterial activity against

K.pneumoniae, P.vulgaris.

Fig 1. Crossandra infundibuliformis

Fig 2. Standard curve of different concentrations (mg/ml) of

gallic acid and their respective optical density at 725nm

Table 1. Preliminary Phytochemical Screening of Flower Extracts of Crossandra infundibuliformis

Tests Chloroform Ethyl Acetate Acetone Aqueous

Carbohydrates - + + -

Proteins - - - -

Amino acids - - - -

Steroids - - - -

Cardiac glycosides - + + -

Flavonoids - + + +

Alkaloids - + - +

Tannins + + + + ‘+’ indicates positive, ‘-’ indicates negative

Table 2. The total phenolic, non-tannin and tannin content present in flower extracts of Crossandhra infundibuliformis

in mg of GAE/gm of extract

S.No Name of the Extract Total Phenolic Content Non-Phenolic Content Tannin Content

1 Chloroform 12.03±0.50 5.20±0.25 6.83±0.25

2 Ethyl Acetate 7.0±0.62 4.3±0.50 2.7±0.12

3 Acetone 13.0±0.78 4.4±0.28 8.6±0.50

4 Aqueous 12.0±0.55 6.6±0.30 5.4±0.25 The data given is Mean ± Standard Deviation, n=3

326 Sowjanya Pulipati. et al. / International Journal of Biological & Pharmaceutical Research. 2014; 5(4): 323-326.

Table 3. Antibacterial activity of flower extracts of Crossandra infundibuliformis against UTI Pathogens

S.No Name of the

Microorganisms

Zone of Inhibition in mm

Chloroform Ethyl acetate Acetone Aqueous Nitrofurantoin

1. 1 E.coli 10.83±0.76 12.5±0.50 16.20±0.25 11.83±0.26 14.5±0.28

2. 2 K.pneumoniae 11.20±0.50 10.83±0.16 12.50±0.30 09.0±0.12 14.6±0.30

3. 3 E.faecalis 12.5±0.52 11.50±0.50 13.23±0.26 10.2±0.52 13.5±0.50

4. 4 P. aeruginosa 10.53±0.50 11.1±0.36 15.63±0.40 10.5±0.50 12.3±0.25

5. 5 P.vulgaris 09.83±0.28 11.76±0.25 13.66±0.30 09.5±0.30 13.2±0.32 The data given is Mean±Standard Deviation, n=3

CONCLUSION

The antibacterial potential of Crossandhra

infundibuliformis flower extracts against UTI pathogens

needs extensive investigation to understand its antibacterial

principles which may allow scientific community to

recommend its use as accessible alternative to synthetic

antibiotics. It was observed that due to the presence of

higher content of tannin in acetone extract, it showed

prominent antibacterial activity against tested UTI

pathogens. In conclusion it is anticipated that the bioactive

compounds in acetone extract of Crossandhra

infundibuliformis can find its potential as a novel

antibacterial agent might be useful to cure infections

caused by uropathogenic strains.

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