Antimicrobial Evaluation of Lipid Extract of Pongamia pinnata Leaves
*Corresponding Author E-mail: sumoraji@rediffmail.com
ABSTRACT
The aim of the present study was to evaluate the antimicrobial activity of selected plant Pongamia pinnata used in ancient days for the treatment of so many skin diseases. Surface lipids of the selected plant were extracted using chloroform. The crude lipid extract was subjected to column chromatography, by using different solvents pentane, 1% ethyl acetate in hexane, 5% ethyl acetate in hexane, chloroform, methanol yielding five fractions from the crude lipid extract. These fractions were tested against bacterial and fungal strains using agar cup bioassay method and broth dilution method. Bacillus subtilis, Bacillus sphearicus, Staphylococcus aureus, Staphylococcus epidermis, Escheritia coli, Pseudomonas aerogenosa, Klebsella pneumoniae, Candida albicans, Candida rugosa, Sacharomyces cerevisiae, Aspergillus niger, Aspergillus flavus were the 12 microbial strains used for the in vitro antimicrobial study. Pentane and 1% ethyl acetate in hexane fractions showed no activity on all microbial strains except S.epidermidis where as all other fractions showed moderate activity on all microbial strains except E.coli and B.subtilis. All the fractions showed greatest activity against S.epidermidis. In case of antifungal assay, pentane and 1% ethyl acetate fractions showed no activity on all fungal strains, where as rest of the fractions showed moderate activity on all fungal strains. This study supports the selected plant showed encouraging results, as it found to contain substances that had antimicrobial activity because of, the isolated compounds of Pongamia pinnata were active against 10 out of 12 microbial strains.
KEYWORDS: Surface lipids, Pongamia pinnata, Antibacterial activity, Antifungal activity.
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 commomnly called as Karanja, is a evergreen plant seen through out India, mainly for its seeds. 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, but to date there has been no report on the composition of the leaf cuticular waxes.
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.4 The cuticular wax also performs the important function of protecting the leaf against bacterial and fungal pathogens. The present work describes the extraction, isolation and biological evaluation of the different components of leaf cuticular wax 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.
Extraction of Cuticular Lipids 5: 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.
Detection and Separation of Cuticular Waxes 6: Different constituents of cuticular lipids were detected by TLC (toluene as mobile phase; detection by iodine vapors and also by spraying with 5% sulphuric acid in methanol followed by charring). Five spots were localized corresponding to hydrocarbons, ketones, aldehydes, primary alcohols and some clusters of spots containing free fatty acids and sterols. Reference samples of octadecane, palmmitone, cetyl alcohol, oleic acid and stigmasterol were spotted alongside for confirmation of different classes of compounds 7. The chromatographic separation of cuticular lipids was carried out quanitatively by silica gel column. Five fractions were eluted with pentane, 1% ethyl acetate in hexane, 5% ethyl acetate in hexane, chloroform and methanol 8.
In vitro Antibacterial assay 9: The antibacterial activity of the six fractions 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 agar cup bioassay 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 chloroform and three different concentrations were made (50μg/ml, 100μg/ml, 150 μg/ml). After inoculation, wells were scooped out with 6mm sterile cork borer and the lids of the dishes were replaced. To each well, different concentrations of test solutions were added separately. Controls were maintained with chloroform 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 antifungal activity of the six fractions 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 agar cup bio assay method. The readymade PDA medium was suspended in distilled water (1000ml) and heated to boiling until it is dissolved completely. The medium 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 acetone and three different concentrations were made (50μg/ml, 100μg/ml, 150 μg/ml). After inoculation, cups were scooped out with 6mm sterile cork borer and the lids of the dishes were replaced. To each cup, different concentrations of test solutions were added separately. Controls were maintained with chloroform 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 10:
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 100µg/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.
Table No. 1 shows the composition of lipid classes from the leaves of Pongamia pinnta
Compound Class |
Content Weight % |
F1 |
4.6 |
F2 |
21.8 |
F3 |
46.0 |
F4 |
6.9 |
F5 |
12.6 |
Table No. 2 shows anti bacterial activity of isolated fractions from the surface lipid extract of Pongamia pinnata
Zone of Inhibition in mm |
||||||||
Compound code |
Concentration |
B.subtilis |
B.sphearicus |
s.aureus |
s.epidermidis |
E.coli |
P.aeroginosa |
K.pneumoniae |
1 |
50 |
0 |
0 |
0 |
8 |
0 |
0 |
0 |
|
100 |
0 |
9 |
0 |
11 |
0 |
0 |
0 |
|
150
|
0 |
10 |
9 |
15 |
0 |
0 |
0 |
2 |
50 |
0 |
0 |
0 |
14 |
0 |
0 |
0 |
|
100 |
0 |
10 |
0 |
17 |
0 |
0 |
0 |
|
150
|
0 |
10 |
10 |
22 |
0 |
0 |
0 |
3 |
50 |
0 |
0 |
0 |
10 |
0 |
0 |
0 |
|
100 |
0 |
11 |
0 |
14 |
0 |
0 |
0 |
|
150
|
0 |
14 |
12 |
17 |
0 |
10 |
0 |
4 |
50 |
0 |
10 |
0 |
8 |
0 |
0 |
0 |
|
100 |
0 |
12 |
9 |
10 |
0 |
0 |
0 |
|
150
|
0 |
12 |
12 |
13 |
0 |
10 |
0 |
5 |
50 |
0 |
10 |
0 |
11 |
0 |
0 |
0 |
|
100 |
0 |
15 |
10 |
15 |
0 |
0 |
8 |
|
150
|
0 |
15 |
15 |
18 |
0 |
15 |
10 |
6 |
50 |
0 |
8 |
0 |
8 |
0 |
0 |
8 |
|
100 |
0 |
10 |
10 |
10 |
0 |
12 |
10 |
|
150
|
0 |
12 |
10 |
12 |
0 |
12 |
10 |
7 |
50µg |
19 |
21 |
21 |
23 |
29 |
24 |
23 |
Table No.3 shows anti fungal activity of isolated fractions from surface lipid extract of Pongamia pinnata
Zone of Inhibition in mm |
||||||
Compound code |
Concentration |
C.albicans |
C.rugosa |
S.cerevisiae |
A.niger |
A.flavus |
1 |
50µg |
0 |
0 |
0 |
0 |
0 |
|
100µg |
0 |
0 |
0 |
0 |
0 |
|
150mg
|
0 |
0 |
0 |
0 |
0 |
2 |
50µg |
0 |
0 |
0 |
0 |
0 |
|
100µg |
0 |
0 |
0 |
0 |
0 |
|
150 mg
|
0 |
0 |
0 |
0 |
0 |
4 |
50µg |
0 |
0 |
0 |
0 |
0 |
|
100mg |
8 |
8 |
0 |
0 |
0 |
|
150µg
|
10 |
8 |
8 |
8 |
8 |
5 |
50mg |
0 |
0 |
0 |
0 |
0 |
|
100µg |
8 |
8 |
8 |
9 |
8 |
|
150µg
|
11 |
11 |
10 |
12.5 |
12 |
6 |
50mg |
0 |
0 |
0 |
0 |
0 |
|
100µg |
7 |
9 |
8 |
8 |
8 |
|
150mg
|
10 |
12 |
12 |
11.5 |
11 |
7 |
50µg |
0 |
0 |
0 |
0 |
0 |
|
100µg |
7 |
10 |
12 |
11 |
13 |
|
150mg
|
8 |
10 |
12 |
12 |
14 |
8 |
50µg |
23.5 |
25 |
22 |
25 |
24 |
Table No.4 shows Minimum Inhibitory Concentration of the isolated fractions from the surface lipid extract of Pongamia pinnata
Organism |
MIC in mg/ml |
|||||
Fractions |
||||||
|
1 |
2 |
3 |
4 |
5 |
6 |
B.sphearicus |
50 |
50 |
50 |
25 |
12.5 |
12.5 |
S.aureus |
50 |
50 |
50 |
50 |
50 |
50 |
S.epidermidis |
6.25 |
6.25 |
6.25 |
6.25 |
6.25 |
3.125 |
P.aeroginosa |
- |
- |
- |
50 |
50 |
50 |
K.pneumoniae |
- |
- |
- |
50 |
50 |
50 |
C.albicans |
- |
- |
- |
50 |
50 |
50 |
C.rugosa |
- |
- |
- |
50 |
50 |
50 |
S.cerevisiae |
- |
- |
- |
50 |
50 |
50 |
A.niger |
- |
- |
50 |
50 |
50 |
50 |
A.flavus |
- |
- |
50 |
50 |
50 |
50 |
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%.
Separation of lipid classes by Column Chromatography: The surface lipids of Pongamia pinnata were separated quantitatively into five fractions using pentane, 1%ethyl acetate in hexane, 5% ethyl acetate in hexane, chloroform and methanol respectively. These fractions were designated as F1, F2, F3, F4, F5 and their composition is given in Table No.1.
Identification of lipid classes: All the fractions collected during column chromatography were subjected to thin layer chromatography for their identification.
Fraction 1: Based on the column chromatography analysis, the content of first fraction was found to be 4.6% of the total surface lipids extracted. The eluted fraction was spotted on a pre-coated TLC plate along with a standard hydrocarbon, octadecane. The TLC plate was run using a solvent system containing toluene. After developing the TLC plates, the solvent was allowed to dry and was sprayed with 5% sulphuric acid and charred on a hot plate to visualize the spots. The Rf of standard hydrocarbon and fraction 1 (0.98) were found to be same. Hence it was concluded that fraction 1 contains hydrocarbons.
Fraction 2: The second fraction eluted with 1% ethyl acetate in hexane solvent system from the column was about 21.8% of total surface lipids extracted. The eluted fraction was spotted on a pre-coated TLC plate along with a standard ketone, palmitone. The TLC plate was run using a solvent system containing toluene. After developing the TLC plates, the solvent was allowed to dry and was sprayed with 5% sulphuric acid and charred on a hot plate to visualize the spots. The Rf of standard ketone and fraction 2 (0.87) were found to be same. Hence it was concluded that fraction 2 contains ketones.
Fraction 3: Based on the column chromatography analysis, the content of third fraction eluted with 5%ethyl acetate in hexane solvent system was found to be 46% of the total surface lipids extracted. The eluted fraction was spotted on a pre-coated TLC plate along with a standard cetyl alcohol. The TLC plate was run using a solvent system containing toluene. After developing the TLC plates, the solvent was allowed to dry and was sprayed with 5% sulphuric acid and charred on a hot plate to visualize the spots. The Rf of standard and fraction 3 (0.70) were found to be same. Hence it was concluded that fraction 3 contains alcohols.
Fraction 4: The fourth fraction eluted with chloroform from the column was about 6.9% of total surface lipids extracted. Qualitatively, the fraction was analyzed by using TLC technique and comparing with pure standard oleic acid. The Rf of standard and fraction 4 (0.34) were found to be same. Hence it was concluded that fraction 4 contains fatty acids.
Fraction 5: The final fraction eluted with methanol from the column was about 12.6% of the total surface lipids extracted. The eluted fraction was spotted on a pre-coated TLC plate along with a standard stigmosterol. The TLC plate was run using a solvent system containing toluene. After developing the TLC plates, the solvent was allowed to dry and was sprayed with 5% sulphuric acid and charred on a hot plate to visualize the spots. The Rf of standard stigmosterol and fraction 5 (0.15) were found to be same. Hence it was concluded that fraction 5 contains sterols.
Antibacterial activity: Five fractions that are eluted from the column i.e., Pentane fraction, 1% ethyl acetate in hexane fraction, 5% ethyl acetate in hexane fraction, Chloroform fraction, Methanol fraction and the crude sample of surface lipids were selected for antibacterial activity. Seven bacterial strains were used for antibacterial screening. Various concentrations of the extracts (50, 100 and 150 µg/ml) were used to test the antibacterial activity. Streptomycin was used as standard drug and Chloroform as negative control. The results were shown in Table No.2. From the results of antibacterial screening, Chloroform and Methanol extracts showed significant activity on all bacterial strains, except B.subtilis and E.coli. Pentane extract showed no activity in all, except B.sphearicus and S.epidermidis. Rest of the extracts S. aureus, P.aeroginosa, K.pneumoniae has medium activity on all bacterial strains. Compounds 1-5 are the fractions eluted with Pentane, 1% Ethylacetate in hexane, 5% ethyl acetate in hexane, Chloroform, methanol respectively, compound 6 is the crude surface lipid extract and Compound 7 is the standard drug. The results were given in Table No.2.
Antifungal activity: Five fractions that are eluted from the column i.e., Pentane fraction, 1% ethyl acetate in hexane fraction, 5% ethyl acetate in hexane fraction, Chloroform fraction, Methanol fraction and the crude sample of surface lipids were selected for antifungal activity. Five fungal strains were used for antifungal screening. Three concentrations of the extracts (50,100 and 150 µg/ml) were used to test the antifungal activity. From the results of antifungal screening, 5% ethyl acetate in hexane extract, Chloroform extract and Methanol extract showed significant activity on C.albicans C.rugosa, S.cerevisiae, A.niger, A.flavus while Pentane extract and 1% ethyl acetate in hexane extract showed no activity. Amphotericin B was used as standard drug and Chloroform as negative control. Compounds 1-5 are the fractions eluted with Pentane, 1% Ethylacetate in hexane, 5% ethyl acetate in hexane, Chloroform, methanol respectively, compound 6 is the crude surface lipid extract and Compound 7 is the standard drug. The results were shown in Table No.3
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.4
CONCLUSION:
The surface lipids were extracted from Pongamia pinnata leaves and the individual groups of components were isolated by column chromatography. In vitro antimicrobial study was carried out by using nutrient agar medium which showed antibacterial activity against the some gram + and gram– bacteria (B.sphearicus, s.aureus, s.epidermidis, P.aeroginosa, K.pneumoniae) and antifungal activity against C.albicans, C.rugosa, S.cerevisiae, A.niger, A.flavus. So from our study it was concluded that the surface lipids extracted and isolated from Pongamia pinnata leaves will be useful for future study.
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Received on 27.03.2009 Modified on 23.05.2009
Accepted on 21.06.2009 © RJPT All right reserved
Research J. Pharm. and Tech.2 (4): Oct.-Dec. 2009; Page 714-718