INTRODUCTION:

The existence of mankind depends on herbal medicines which are the gift of nature. It has been universally accepted that plant drugs and remedies are safer than synthetic medicines even for curing complex diseases. Extraction of bioactive compounds such as tannins facilitates synthesis of potent drugs with reduced toxicity. Tannins are polyphenols that are obtained from various parts of plants. It is used in pharmaceutical, food and leather industries as well as in the agricultural sector. According to many studies tannins have shown potential antibacterial, antiviral and antiparasitic effects^1-7.

The various microorganisms that cause urinary tract infection are Escherichia coli, Proteus mirabilis, Staphylococcus saprophyticus and Klebsiella. The synthetic drugs available for treating urinary tract infection have side effects and drug resistant strains are also emerging. Therefore herbal medicines are safe.

Herpestis monniera (Linn) belongs to Scrophulariaceae. It is a prostrate, juicy, succulent, glabrous, annual herb rooting at the nodes with numerous ascending branches. Leaves are simple, opposite, decussate, sessile, obovate, oblong or spatulate entire, fleshy, obscurely veined and punctate. Flowers are pale blue or whitish, axcillary, solitary, arranged as long and slender pedicels. Fruits are ovoid, acute, two celled; two valved capsules and tipped with style base. Seeds are minute and numerous⁸.

MATERIALS AND METHODS:

Microorganisms:

Clinical isolates of the bacteria namely Escherichia coli, Proteus mirabilis, Staphylococcus Saprophyticus and Klebsiella were obtained from Government Hospital, Vellore.

Source of plant material:

The leaves of the plant were collected from Kancheepuram, Tamilnadu and the species was duly authenticated at Botanical Survey of India, Coimbatore, Tamilnadu.

Preparation of plant extract:

Plant materials were collected, shaded, dried and 50gms of powdered sample was used. The blended material was transferred into a beaker and 200ml of solvent (Methanol: Water- 9:1) added at ambient temperature (28+/- 2°C). The mixture was extracted by agitation on a rotary shaker. Extraction was allowed to proceed for 48 hours. The extract was decanted and the solvent was removed by evaporation at ambient temperature (28+/- 2°C) to obtain the extract. The air dried extract was stored for 48hours in sterile bottles at room temperature⁹.

Extraction of tannins and phytochemical screening:

Sample of the powdered leaf (3g) was boiled in 5ml of distilled water for 3 minutes on heating mantle. The mixture was filtered while hot and the resulting filtrate was used to carry out ferric chloride test. Sample of the filtrate (1.0gm) was transferred into a beaker and 10ml of distilled water was added and boiled for 5minutes. Two drops of 5 percent ferric chloride was added. Production of greenish precipitate indicated the presence of tannins¹⁰.

Table 1: Anti Bacterial Activity of Tannin

Concentration (mg/ml)	Escherichia coli	Poteus mirabilis	Staphylococcus saprophyticus	Klebsiella
0.5	Nil	Nil	Nil	Nil
1.0	Nil	Nil	Nil	Nil
1.5	Nil	8.3	Nil	9.6
2.0	7.5	11.7	7.3	10.7
2.5	9.8	12.8	9.1	11.9

Fig 1:Herpestes monnieria (L.) Kunth

Separation of tannins using thin layer chromatography:

The plates were prepared and spotted with crude tannin extracts. The spotted plates were then placed in TLC tank containing the solvent system Hexane: Ethylacetate: Methanol in the ratio of 40:40:20. The plates were then sprayed with ferric chloride reagent and greenish precipitate showed the presence of tannins.

Antimicrobial activity:

A modified antibacterial test was performed using the agar diffusion method¹¹. The microorganisms were inoculated on Muller Hinton agar (MHA) and spread uniformly using a sterile spreader. Wells of 5mm in diameter were made on MHA using a sterile well puncher. The cut agar blocks were carefully removed by the use of forceps sterilized by flaming. Different concentrations (0.5, 1.0, 1.5, 2.0, 2.5mg/ml) of tannins isolated from the plant extract. Controls were maintained. The plates were allowed to stand for one hour at room temperature for diffusion of the substances before the growth of the microorganism commenced. The plates were incubated at 37 C for the zone of inhibition was recorded.

Table 2: Minimum Inhibitory Concentration and Minimum Bactericidal Concentration of Tannins

Test Organism	MIC (mg/ml)	MBC (mg/ml)
Escherichia coli	4.5	5.0
Poteus mirabilis	4.0	4.5
Staphylococcus saprophyticus	4.5	5.0
Klebsiella	3.5	4.0

Fig. 2: Basic Structure of a Tannin - Tannins consist mainly of gallic acid residues that are linked to glucose via glycosidic bonds.

Determination of Minimum Inhibitory Concentration (MIC):

Various concentrations of tannins from the plant Herpestis monniera ranging between 3-5mg/ml were introduced into different test tubes. Each tube was inoculated with an overnight culture of Escherichia coli, Proteus mirabilis, Staphylococcus Saprophyticus and Klebsiella diluted to give a final concentration of 106 cells per ml. The tubes were incubated at 37 C for 24 hours. The least concentration of tannin that did not permit any visible growth of the inoculated test organism in broth culture was regarded as minimum inhibitory concentration in each case¹¹.

Determination of Minimum Bactericidal Concentration (MBC):

After culturing the test organism separately in the nutrient broth containing various concentrations of the active ingredients, the broth was inoculated onto freshly prepared agar plates to assay for bactericidal effect. The culture was incubated at 37 C for 24 hours. The lowest concentration of tannin that does not yield any colony growth on the solid medium after the incubation period was regarded as minimum bactericidal concentration¹².

RESULTS AND DISCUSSION:

Tannins isolated from Herpestis monniera in this study exhibited anti bacterial activity against Escherichia coli, Proteus mirabilis, Staphylococcus saprophyticus and Klebsiella shown in Table 1. The isolated tannins from herbal extracts exhibited high anti bacterial activity against all the pathogens. However the highest effect was on Proteus mirabilis followed by Klebsiella, Escherichia coli and Staphylococcus Saprophyticus at 2.5 mg/ml. The plants that are rich in tannins possess antimicrobial activities¹³.

MIC of tannins isolated in this study against the test organisms ranged between 3.0 and 4.5 mg/ml while the MBC ranged between 3.5 and 5.0 mg/ml (Table 2). The anti bacterial effectiveness observed with increase in concentration of tannins observed in the present study is in agreement with the earlier investigations¹⁴.

CONCLUSION:

A high concentration of tannin can cure UTI caused by the following microorganisms-Escherichia coli, Proteus mirabilis, Staphylococcus saprophyticus and Klebsiella. The antibacterial effectiveness of the tannin indicates the possibility of developing the plant as a source of anti microbial agent. The present study clearly supports the traditional use of Herpestis monniera against all urinary tract infections.

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Received on 11.02.2010 Modified on 28.02.2010

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 832-834