INTRODUCTION:

Herbal medicines are the oldest remedies known to mankind herbs had been used by all cultures throughout history. Plant extracts and phytochemicals are becoming popular as potential source of antibacterials, antifungals and antivirals, and several reviews have been written.^[1]But India has one of the oldest, richest and most diverse cultural living traditions associated with use of medicinal plants. Experimentation with plants and passage of knowledge from one generation to next resulted in the development of a vast knowledge about plants to use as medicines and narcotics.^[2]In the present scenario, the demand for herbal products is growing exponentially throughout the world and pharmaceutical companies are currently conducting extensive research on plant materials for their potential medicine value. Bacterial, fungal and viral infections are important health problems all over the world, both in developed and developing countries, due to morbidity and mortality ^[3].

In many national and international journals, we find an interesting number of research publications based on herbal drugs and their formulations. Antibiotic therapy is an essential strategy for treatment of hazardous microbial diseases; however its efficiency is restricted by both natural and acquired cell resistances to drugs. Antibiotic compounds with new cellular targets are needed. To overcome the above circumstances, antimicrobial compounds which minimize the side effects and increase the efficiency of antibiotic therapy have to be investigated and isolated. Several plant parts described as potential antibacterial and antifungal agents have been reported. The selected plant Pongamia pinnata belongs to family Leguminosae and is commonly called as Karanja, is a evergreen plant seen throughout India, mainly for its seeds^[4]. The medicinal importance of this plant has led many investigators to study in detail regarding the uses. There were reports on the whole extracts of leaves citing the antiviral, antibacterial, antidiarrheal activity ^[5].

The cuticular wax covering the leaf cuticle performs many diverse biological functions, which are vital for plant life. It restricts non-stomatal water loss, thus maintaining the water balance of the plant and it reduces water retention on the leaf, keeping the leaf surface dry and thus preventing the germination of pathogen spores and minimizing deposition of dust, pollen and air pollutants. The cuticular wax also performs the important function of protecting the leaf against bacterial and fungal pathogens ^[6]. The present work describes the extraction, biological evaluation and comparison of leaf cuticular waxes and total lipids of Pongamia pinnata.

MATERIALS AND METHODS:

Collection of sample:

The leaves were collected from Pongamia pinnata tree from Indian Institute of Chemical Technology campus, Hyderabad. The plant was identified and authenticated by Taxonomist from Department of Botany, Osmania University, Hyderabad.

Extraction of Cuticular Lipids^[7]:

Cuticular lipids were extracted from fresh leaves by immersing consecutively into two 500ml beakers of chloroform for a total of 1min. During the extraction, care was taken to avoid immersing any damaged or cut leaves in the solvent. The extraction was repeated in triplicate using fresh leaves. The combined chloroform extracts were then filtered dried over Na₂SO₄ and concentrated using rotary evaporator under reduced pressure.

Extraction and Purification of Total Lipids ^[8]:

The leaves of Pongamia pinnata were collected and dried under shade. Dried leaves were crushed into powder in a mortor. The grounded powder was mixed with a solvent mixture consisting of chloroform and methanol (1:2 v/v) and magnetically stirred for 6-12 hrs. The obtained organic residue was dried over anhydrous sodium sulphate and concentrated at 60-70°C using rotary evaporator.

Removal of Chlorophyll Pigment from the Total Lipids: The extracted total lipids contain considerable amounts of chlorophyll and other pigments. The extract is added to chloroform solution and was applied in a short column (1.2 cm. i.d.) of a mixture of activated charcoal and celite 545 (2:1 w/w).

In vitro Antibacterial Activity ^[9]:

The comparative antibacterial activity of surface and total lipids was studied against seven bacterial strains. The test organisms, Bacillus subtilis (MTCC 441), Bacillus sphearicus (MTCC 511), Staphylococcus aureus (MTCC 96), Staphylococcus epidermis (MTCC 817), Escheritia coli (MTCC 523), Pseudomonas aerogenosa (MTCC 741), Klebsella pneumoniae (MTCC 59), were obtained from the Institute of Microbial Technology, Chandigarh, India. Cultures of test organisms were maintained on nutrient agar slants and were sub cultured in Petri dishes prior to testing. Three replicates were maintained for each treatment. The antibacterial activity was performed using paper disc method. Nutrient agar medium was weighed and suspended in distilled water (1000ml) and heated to boiling until it is dissolved completely. The medium was autoclaved at a pressure of 15 lb/inc² for 20 min. The medium was poured into sterile Petri dishes under aseptic conditions in a laminar flow chamber. When the medium in the plates solidified, 0.5 ml of test culture was inoculated and uniformly spread over the agar surface with a sterile L-shaped bent glass rod. Solutions were prepared by dissolving the test compounds in di methyl sulfoxide (DMSO) and two different concentrations^[110] were made (100μg/ml, 150 μg/ml). After inoculation, filter paper discs soaked in test compounds and controls were placed aseptically on the surface of the solidified nutrient agar. Controls were maintained with DMSO and streptomycin (50μg/ml). The treated and the controls were incubated at room temperature for 48 hr. Inhibition zones were measured and diameter was calculated in millimeter. Three replicates were maintained for each treatment.

In vitro Antifungal assay ^[10]:

The comparative antifungal activity of surface and total lipids was studied against five fungal strains. These strains, Candida albicans (MTCC 2656), Candida rugosa (MTCC 262), Sacharomyces cerevisiae (MTCC 984), Aspergillus niger (MTCC 282), Aspergillus flavus (MTCC 2030) were obtained from the Institute of Microbial Technology, Chandigarh, India. Cultures of test organisms were maintained on Potato dextrose Agar (PDA) slants and were sub cultured in petri dishes prior to testing for antifungal activity using paper disc method. The readymade PDA medium was suspended in distilled water (1000ml) and heated to boiling until it is dissolved completely. The medium was autoclaved at a pressure of 15 lb/inc² for 20 min. The medium was poured into sterile Petri dishes under aseptic conditions in a laminar flow chamber. When the medium in the plates solidified, 0.5ml of test culture was inoculated and uniformly spread over the agar surface with a sterile L-shaped bent glass rod. Solutions were prepared by dissolving the test compounds in di methyl sulfoxide (DMSO) and two different concentrations^[110] were made (100μg/ml, 150 μg/ml). After inoculation, filter paper discs soaked in test compounds and controls were placed aseptically on the surface of the solidified potato dextrose agar. Controls were maintained with DMSO and amphoterecin-B (50μg/ml). The treated and the controls were kept at room temperature for 48 hr. Inhibition zones were measured and diameter was calculated in millimeter. Three replicates were maintained for each treatment.

Minimum inhibitory concentration of isolated compound ^[9]:

1) A series of culture tubes were prepared all containing the same volume of medium inoculated with test microorganisms. The lowest concentration of sample at which the subculture from test dilution yielded no viable organisms was recorded as minimum inhibitory concentration.

2) Decreasing concentration of drug was added to the tubes usually a step wise dilution (two fold serial dilutions) was used starting from 200µg/ml to 1.5625µg/ml. One tube was left without drug to serve as positive control and other without drug and inoculum to serve as negative control.

3) The cultures were incubated at a temperature optimal for growth of the test organism and a period of time sufficient for growth for at least 10-15 generators (usually 24hrs for bacteria at 37°C and 48 hrs for fungi at 28°C).

4) The tubes were inspected visually to determine the growth of organisms by the presence of turbidity and the tubes in which antibiotic is present in minimum concentration sufficient to inhibit the microbial growth which remains clear was noted as MIC of the extracts.

5) In experimental terms MIC is the concentration of the drug present in the last clear tube, i.e. the tube having the lowest antibiotic concentration in which growth is not observed.

RESULT AND DISCUSSION:

Lipid content:

The surface lipids were extracted by dipping the leaves in chloroform over a period of 1min. The amount of surface lipids was found to be 0.12%. Total lipids were extracted by using chloroform and methanol solvent mixture, and the amount was found to be 1.7%.

Antibacterial activity:

Crude samples of surface lipids and total lipids were selected for antibacterial activity. Seven bacterial strains were used for antibacterial screening. Two concentrations of the extracts (100 and 150 µg/ml) were used to test the antibacterial activity. Streptomycin was used as standard drug and DMSO as negative control. The results were shown in Table No.1and Fig 1. From the results of antibacterial screening, surface lipid extract showed significant activity on all bacterial strains, except B.subtilis and E.coli. Total lipid extract showed less activity in high concentration.

Fig.1: Comparative Anti-bacterial Activity of Surface and Total lipids

Antifungal activity:

Crude sample of surface lipids and total lipids were selected for antifungal activity. Five fungal strains were used for antifungal screening. Two concentrations of the extracts (100 and 150 µg/ml) were used to test the antifungal activity. From the results of antifungal screening, it was found that, both the extracts showed significant activity on all fungal stains. But the total lipid extract was found less active when compared with surface lipids. The results were shown in Table No.2 and Fig 2.

Fig.2 Comparative Anti-fungal Activity of Surface and Total lipids

Table No:1 Comparative anti bacterial activity of Ponagamia pinnata leaf surface and total lipid extracts.

Zone of Inhibition in mm
Compound Name	Concentration (µg/ml)	*B.subtilis*	*B.sphearicus*	*S.aureus*	*S.epidermidis*	*E.coli*	*P.aeroginosa*	*K.pneumoniae*
Surface Lipids	100	0	12	10	14	0	10	8
	150	0	15	12	17	0	15	10
Total lipids	100	0	0	0	0	0	0	0
	150	0	5	7	8	0	10	6
Standard	50	19	21	21	23	22	19	22
Control		0	0	0	0	0	0	0

Table No: 2 Comparative anti fungal activity of Ponagamia pinnata leaf surface and total lipid extracts.

Zone of Inhibition in mm
Compound Name	Concentration (µg/ml)	*C.albicans*	*C.rugosa*	*S.cerevisiae*	*A.niger*	*A.flavus*
Surface Lipids	100	8	9	8	9	13
	150	11	12	12	12.5	14
Total lipids	100	5	6	0	6	0
	150	8	8	9	10	7
Standard	50	18	21	22	24	22
Control		0	0	0	0	0

Minimum Inhibitory Concentration:

The Minimum Inhibitory Concentration was evaluated to know the lowest antibiotic concentration in which growth was not observed. The values for different test organisms were given in Table No.3

Table No.3 Minimum Inhibitory Concentration of the extracted compounds from Pongamia pinnata leaves

Organism	MIC in mg/ml
	Fractions
	Surface lipids	Total lipids	Standard	Control
B.sphearicus	50	100	50	0
S.aureus	25	200	50	0
S.epidermidis	6.25	100	6.25	0
P.aeroginosa	25	200	12.5	0
K.pneumoniae	50	100	25	0
C.albicans	25	50	12.5	0
C.rugosa	12.5	50	6.25	0
S.cerevisiae	25	100	6.25	0
A.niger	25	50	12.5	0
A.flavus	50	50	25	0

CONCLUSION:

The surface lipids and total lipids were extracted from Pongamia pinnata leaves and the crude extracts were evaluated for comparative anti-microbial study. In anti-bacterial evaluation, 5 out of 7 strains responded to the extracts showing zone of inhibition. In anti-fungal evaluation, all the fungal strains responded to the extracts showing zone of inhibition. In the comparative study, it was observed that the activity of surface lipids was more. This study supports that the selected plant showed encouraging results, as it found to contain substances that has antimicrobial activity. So from our study it was concluded that the surface lipids extracted from Pongamia pinnata leaves will be useful for future study.

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Received on 09.09.2014 Modified on 25.10.2014

Res. J. Pharmacognosy & Phytochem. 7(1): Jan.-Mar. 2015; Page 25-28

DOI: 10.5958/0975-4385.2015.00006.0

Pavani. S*, Dr. Rao. B.V.S.K, Dr. M. Srinivas Murthy

Department of Pharmaceutical Biotechnology.

Vignan Institute of Pharmaceutical Sciences, Deshmukhi, Nalgonda, Telangana-508284, India.