Antimicrobial Activity of Polyalthia longifolia ( Sonn . ) Thw . var . Pendula Leaf Extracts Against 91 Clinically Important Pathogenic Microbial Strains

The methanol, acetone and 1,4-dioxan fractions of leaves of Polyalthia longifolia (Sonn.) Thw. were evaluated for antibacterial and antifungal activity. 91 clinically important strains were used for the study which were both clinical isolates as well as identified strains. Piperacillin and gentamicin were used as standards for antibacterial assay, while nystatin and flucanazole were used as standards for antifungal assay. The antibacterial activity was more pronounced against gram positive bacterial and fungal strains. Poor activity was shown against gram negative bacterial strains studied.


Introduction
Due to the increasing development of drug resistance in human pathogens as well as the appearance of undesirable effect on certain antimicrobial agents, there is a need to search for new agents.The world health organization in 1997 suggested that effective locally available plants be used as substitutes for drugs.Research work on medicinal plants be intensified and information on these plants be exchanged.This thought will go a long way in the scientific exploration of medicinal plants for the benefit of man and is likely to decrease the dependence on importance of drugs [1].Polyalthia longifolia (Annonaceae) is a tree, which is widely distributed in Bangladesh, Srilanka and throughout the hotter parts of India [2].In India, the seeds of this plant were used as febrifuge [3].Literature survey revealed that most of the plants of annonaceae family contain antitumor and anticancer principles [4,5].The bark is also used as a febrifuge in the Balasore district of Orissa [6].The extract of stem bark and the alkaloids isolated from this were found to demonstrate a good antibacterial and antifungal activities [7].In the present study, antimicrobial potentiality of the P. longifolia leaves was investigated against a few clinically isolated as well as standard microbial cultures.

Plant Material
Polyalthia longifolia (Sonn.)Thw.(Annonaceae) leaves were collected in May, 2004 from Rajkot in the State of Gujarat (Western India) and identified by comparison with specimens (PSN 4) available at the Herbarium of the Department of Biosciences, Saurashtra University, Rajkot, Gujarat, India.

Extraction
Leaves of P. longifolia were collected, air dried and then powdered in a homogenizer and 10 gm was used for different solvent extractions (Methanol, Acetone, N, N-dimethylformamide); the sample was extracted in solvent kept on a rotary shaker overnight, and then the filtrate was collected and centrifuged at 5000 rpm.The solvent was then evaporated to dryness under reduced pressure and the extracted compound left was used for the antimicrobial assay.The percentage yield of 1, 4-dioxan, methanol and acetone extracts were 20.56, 29.30 and 13.52 respectively.
Microorganisms Studied 91 clinically important microbial strains which included 23 gram positive, 56 gram negative and 15 fungal strains were studied for the antimicrobial activity.These strains included both clinical isolates as well as identified strains.The details of the microorganisms used are shown in Table 1.

Preparation of Samples
Methanol, acetone and 1,4-dioxan extracts were dissolved in 100% DMSO at a concentration of 25 mg/ml and 12.5 mg/ml and were used as working stocks respectively.Sterile discs (Hi-media Labs) were impregnated with 20 µl of the stock solution.Gentamicin (10 µg/disc) and Piperacillin (100 µg/disc) for bacteria; nystatin (100 units/disc) and flucanazole (10 µg/disc) (Himedia Labs) for fungus were used as positive control and pure DMSO was used as a negative control.

Antimicrobial Study
Antimicrobial activity was performed by agar disc diffusion method [8,9].The bacterial strains were grown in nutrient broth while fungal strains were grown in MGYP (Malt glucose yeast peptone) broth.Mueller Hinton agar no. 2 was the media used to study the antibacterial susceptibility while Sabroaud agar was used to study the antifungal susceptibility test.The cultures were grown for 24 hours, and the turbidity of the culture was maintained according to the 0.5 MacFarland standards.The inoculum's size was 1 × 10 8 cells/ml.

Agar Disc Diffusion
The media (Mueller Hinton Agar No.2 and MRS media) and the test bacterial cultures were poured into Petri dishes (Hi-Media).The test strain (200 µl) was inoculated into the media (inoculums size 10 8 cells/ml) when the temperature reached 40-42°C.The test compound (20 µl) was impregnated in to sterile discs (7 mm) (Hi-Media) and was then allowed to dry.The disc was then introduced into medium with the bacteria.The plates were incubated overnight at 37°C for bacterial strains and 28°C for fungal strains.The experiment was performed under strictly aseptic conditions.Microbial growth was determined by measuring the diameter of the zone of inhibition.The experiment was performed in triplicates and the mean values of the result are shown in Table 2.

Results and Discussion
Herbal medicine in developing countries is commonly used for the traditional treatment of health problems [10].
In recent years multiple drug resistance in human pathogenic microorganisms has developed due to the indiscriminate use of commercial antimicrobial drugs commonly used in the treatment of infectious diseases, making it a global growing problem [11][12][13].In addition to this problem antibiotics are sometimes associated with adverse effects on host including hypersensitivity, immune suppression and allergic reactions [14].Therefore there is a need to develop alternative antimicrobial drugs for the treatment of infections obtained from various sources such as medicinal plants [15,16]   health problem worldwide.Salmonella infection is primarily associated with gastroenteritis.This illness poses a more serious health risk to sensitive populations in the community such as the elderly, young and the immunocompromised, where hospitalization may be required.
All the three extracts were inactive against E. coli, A. fecalis and S. typhimurium.Several antimycotic drugs are available at present, its use is limited by a number of factors such as low potency, poor solubility, emergence of resistance strains and drug toxicity.Therefore there is distinct need for the discovery of new, safer and more effective antifungal agents.Candida species have become a common cause of hospital acquired infections and a large number of patients die as a result of invasive Candidal infections [18].All the three extracts were active against 62.5% of the total fungal strains studied.The three extracts were active against A. candidus while it was inactive against the remaining two moulds (A.flavus and A. niger) studied.The details of the results are given elaborately in Table 2. From the results obtained, it seems that the antibacterial action of the extracts is more pronounced on gram positive than on gram negative bacteria and these findings correlate with the observations of previous screenings of medicinal plants for antimicrobial activity, where most of the active plants showed activity against gram positive strains only [19][20][21].This difference in susceptibility is because of the difference in cell wall structure of gram positive and gram negative organisms.The lipopolysaccharide content of gram negative bacteria makes them resistant to plant extracts while the peptidoglycan layer of gram positive bacteria is not an effective permeability barrier.

Conclusions
All the extracts of P. longifolia exhibited the highest rates of antimicrobial activity against gram positive and fungal strains studied.Therefore, it is concluded that P. longifolia extracts should further be studied phytochemically to elucidate the active principle in the leaf, which can be used as a leading antibacterial (specific for gram positive) and antifungal agent.

Table 1 . List of bacterial and fungal strains studied for an- timicrobial assay.
[17]the three extracts (PDE, PME and PAE at 500 µg/disc concentration) were active against 95% of the total gram positive bacterial strains studied.PDE was active against 18.18% of the total gram negative bacterial strains studied (active against 21% of Pseudomonas spps., 33.3% of Enterobacter spps., 16% of Klebsiella spps., 33.3% of Proteus spps.and66.6% of Citrobacter spps.).PME and PAE were active against 12.72% of the total gram negative bacterial strains studied.P. aeruginosa is most common pathogen of immuno-compromised individuals[17].Infections caused by Pseudomonas spps.are among the most difficult to treat with conventional antibiotics.Both PME and PAE were active against 5.26% of the Pseudomonas spps.
. In the present study, P. longifolia leaf extracts extracted in 1, 4-dioxan (PDE), methanol (PME) and acetone extracts (PAE) were investigated at two different concentrations for their antimicrobial potentiality against 91 clinically important microbial strains.and 66.6% of Enterobacter spps.PME was active against 33.3% of Klebsiella spps.and Proteus spps., while PAE was active against 66.6% of Klebsiella spps.and Proteus spps.studied.Salmonellosis is an important public