Antimicrobial Activity of the Leaves Extract of Punica granatum Linn.

Punasiya R*,Joshi A, Yadav S, Patidar K and Kapse K

GRY Institute of Pharmacy Borawan, Khargone (M.P.) 451228

ABSTRACT:

Human and veterinary medicines have not been so well succeeded in order to achieve their goals concerned with the treatment of various types of infections. The antibiotic activity of Punica granatum Linn. (Fresh leaves) extract was evaluated by the agar diffusion method and agar disc diffusion to determine the zone of inhibition. The extract of P. granatum presented potential antibiotic action over all the assayed strains, forming 10 to 36 mm diameter inhibition zones. This paper’s results claim the effectiveness of the extract of P. granatum as a potential antibacterial agent and display the significance of evaluating new substances with antimicrobial potential, which can contribute to alternative therapeutics for medicine.

KEYWORDS: Punica granatum, aqueous extract, methanol extract and antibiotic activity.

INTRODUCTION:

The antibiotic therapy is the most employed procedure in terms of treatment of various infections to decrease or eliminate the dieses condition.^[1] The increasing failure of chemotherapeutics and antibiotic resistance exhibited by pathogenic microbial infectious agents has bled to the screening of several medicinal plants for their potential antimicrobial activity.^2,3

The rate of resistance to these drugs is higher in developing countries when compared with developed countries. This may be due to the indiscriminate use of antibiotics and also self medications without prescription by physician.⁴

The abusive and indiscriminate use of antibiotic agents in both medical and veterinarian practice have a bottom-up effectiveness regarding the resistance of drugs arising and maintenance.^5,6

Plants with therapeutic properties have a great relevance in medicine throughout the world.^7-9 Punica granatum Linn. Belongs to the Punicacea family, It has a yellowish rind and dark spots, and contains seeds in its core. They are sweet and astringent .^10,11 This shrub is native of northeast India, and it is cultivated all over the world, in tropical and subtropical regions.¹² In Northeast region of Brazil this plant have been used as gargling against infections and inflammations of the tract respiratory.^13,14 Extracts of leaves have exhibited antibacterial activity.¹⁵ and might be used as an effective antibacterial alternative agent against oral biofilm bacteria.¹⁶ The purpose of this survey is to determine the antibiotic activity of the Punica granatum Linn. Fresh leaves extract on bacterial strains. In recent years, secondary plant metabolites (phytochemicals), previously with unknown pharmacological activities, have been extensively investigated as a source of medicinal agents^3,17 The use of plant extracts and phytochemicals, both with known antimicrobial properties, can be of great significance in therapeutic treatments. In the last few years, a number of studies have been conducted in different countries to prove such efficiency¹⁸

MATERIAL AND METHODS:

Collection and Identification

Collection and identification of Plant material fresh plant/plant parts were collected randomly from the West Nimar region of Madhya Pradesh India.

The taxonomic identities of these plants were confirmed by Dr. S.K. Mahajan formerly professor of Govt. P.G. College Khargone (M.P.). Fresh plant material was washed under running tap water, air dried and then homogenized to fine powder and stored in airtight bottles.¹⁶

Preliminary Phytochemical Analysis:

Qualitative phytochemical analysis of the crude powder of Punica granatum plant collected was determined as follows: Tannins (200 mg plant material in 10 ml distilled water, filtered); a 2 ml filtrate + 2 ml FeCl3, blue-black precipitate indicated the presence of Tannins. Alkaloids (200 mg plant material in 10 ml methanol, filtered); a 2 ml filtrate + 1% HCl + steam, 1 ml filtrate + 6 drops of MayorÕs reagents/WagnerÕs reagent/Dragendroff reagent, creamish precipitate/brownish-red precipitate/orange precipitate indicated the presence of respective alkaloids. Saponins (frothing test: 0.5 ml filtrate + 5 ml distilled water); frothing persistence indicated presence of saponins. Cardiac glycosides (Keller-Kiliani test: 2 ml filtrate + 1 ml glacial acetic acid + FeCl3 + conc. H2SO4); green-blue color indicated the presence of cardiac glycosides. Steroids (Liebermann-Burchard reaction: 200 mg plant material in 10 ml chloroform, filtered); a 2 ml filtrate + 2 ml acetic anhydride + conc. H2SO4 Blue-green ring indicated the presence of terpenoids. Flavonoids (200 mg plant material in 10 ml ethanol, filtered); a 2 ml filtrate + conc. HCl + magnesium ribbon pink-tomato red color indicated the presence of flavonoids.^2,19

Extraction of Plant Material:

Aqueous extraction:

10 g of air-dried powder was added to distilled water and boiled on slow heat for 2 h. It was then filtered through 8 layers of muslin cloth and centrifuged at 5000g for 10 min. The supernatant was collected. This procedure was repeated twice. After 6 hours, the supernatant collected at an interval of every 2 hours was pooled together and concentrated to make the final volume one-fourth of the original volume. It was then autoclaved at 121 ^oC and at 15 lbs pressure and stored at 4 ^oC ^{[2,
20, 21, 22]}

Solvent extraction:

10 g of air-dried powder was taken in 100 ml of methanol in a conical flask, plugged with cotton wool and then kept on a rotary shaker at 190-220 rpm for 24 h. After 24 hours the supernatant was collected and the solvent was evaporated to make the final volume one fourth of the original volume and stored at 4 ^oC in airtight bottles.^2,20,22

Bacterial strains:

In vitro antimicrobial activity was examined for aqueous and methanol extracts from Punica granatum. Microorganisms were obtained from the MTCC Institute of Microbial Technology Chandigarh, India. Amongst four microorganisms investigated, two Gram-positive bacteria were Bacillus cereus MTCC430 and Staphylococcus aureus MTCC3160, while two Gram-negative bacteria were Escherichia coli MTCC433 and Klebsiella pneumoniae MTCC432. All the microorganisms were maintained at 4 ⁰C on nutrient agar slants.

Evaluation of antimicrobial activity:

Inoculum Preparation:

0.2 ml of overnight grown cultures of each organism was dispensed into 20 ml of sterile nutrient broth and incubated for 3-5 hr to standardize the culture to 10⁶ CFU/ml.²²

Disk Diffusion method:

10 ul plant extract (concentration 50mg/ml) was soaked by sterile filter paper discs (5mm in diameter). The sterile filter paper disc were impregnated with 10 ul of the plant extract placed on the surface of the medium and incubated at 37^oC for 24 hr. The assessment of antibacterial activity was based on the measurement of diameter of the inhibition zone formed around the disc.^2.22

Agar diffusion method:

The agar diffusion method as described by Esimone et al. (1998) was adopted for the study. 15 ml of molten nutrient agar was seeded with 1.0 ml of standardized broth cultures of the bacteria (1.0 x 10⁷cfu/ml) by introducing the broth cultures into sterile Petri dishes, incorporating the molten agar, rotating slowly to ensure uniform distribution of the microorganisms and then allowed to solidify on a flat surface. Three holes were made in the plates (about 5.0 mm diameter) using a sterile cork borer and equal volumes of the extracts were transferred into the holes using a Pasteur’s pipette. Two Petri dishes containing a particular microorganism were used for each concentration of the extract. The plates were allowed to stand for one hour for prediffusion of the extract to occur and were incubated at 37^oC for 24 hrs.

At the end of incubation the plates were collected and zones of inhibition that developed were measured. The average of the zones of inhibition was calculated. The minimum inhibitory concentration (MIC) was calculated by plotting the natural logarithm of the concentration of extract against the square of zones of inhibition. A regression line was drawn through the points. The antilogarithm of the intercept on the logarithm of concentration axis gave the MIC values.²¹

RESULTS AND DISCUSSION:

Phytochemical Analysis:

Preliminary phytochemical screening revealed the presence of various phytoconstituents given in table.(table-1)

Table No. 1 Phytochemical Analysis

S No.

Phytochemical Analysis

Result

Tannins

Saponin

Flavonoids

Steroids

Cardiac Glycosides

Alkaloids

Terpinoids

+ve

-ve

+ve

The five deferent solvents used for the extraction of the leaves of P. granatum. The antibacterial activity of P. granatum extract was assayed in-vitro by agar disc diffusion and agar well diffusion method against four bacterial species Bacillus cereus (MTCC 430), Staphylococcus aureus (MTCC 3160), Escherichia coli (MTCC 433), Klebsiella pneumoniae (MTCC 432). The antibacterial activity of extracts and their potency was assessed by the presence of zone of inhibition. Table No. 2 summarizes the microbial growth inhibition of aqueous, methanol, ethanol, acetone, chloroform extract of the P. granatum.

Table No. 2 Zone of inhibition of leaves extract of Punica granatum Linn

S. No.	Bacterial Strain	Zone Of Inhibition
S. No.	Bacterial Strain	Chlorophenicol	Aqueous Extract	Methanol Extract	Ethanol Extract	Acetone Extract	Chloroform Extract
01	Bacilus Cereus (MTCC 430)	26	----	12.5	12	13.5	16.5
02	Staphylococcus aureus (MTCC 3160)	24	----	----	----	12.5	13
03	Escherichia Coli (MTCC 433)	18	----	10.2	10.5	11.5	13.5
04	Klebsiella pneumoniae (MTCC 432)	16	----	10.2	9.2	10.5	11.5

The phytochemical analysis of different extract of leaves of P. granatum revealed the presence of tannins, saponins, flavonoids, glycosides, alkaloids, terpinoids (Result shown in table no.1)

The result obtained from antibacterial activity of P. granatum in different solvent was shown in table 2. All the organic solvent extract showed antibacterial activity while aqueous extract did not show zone of inhibition against any of the bacterial strains under studied. Among the five extract tested, chloroform extract showed the better antibacterial activity against all the bacterial strains. The most susceptible bacteria were Bacillus cereus (MTCC 430) while Klebsiella pneumoniae (MTCC 432) was the most resistance strains.

The results of this study support the traditional use of P. granatum. Hence it is necessary for isolation and identification of the compounds of P. granatum extract responsible for antibacterial activity.

CONCLUSION:

The chloroform extract of Punica granatum show maximum antibacterial activity compare with other extract of P.granatum. Aqueous extract did not show zone of inhibition against any of the bacteria strain. Bacillus Cereus (MTCC-430) was most susceptible bacteria while klebsiella pneumoniae (MTCC-432) was the most resistant bacteria.

This investigation is to evaluate the role of P. granatum in bacterial infection (pathogenic condition) and the leaves extract of Punica granatum show antibacterial activity.

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Received on 24.11.2009

Accepted on 30.12.2009

Research Journal of Pharmacognosy and Phytochemistry. 2(1): Jan.-Feb. 2010, 21-24