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Antibacterial and Antifungal Activity of Lectin from Seeds of Pongamia Glabra

Antibacterial and Antifungal Activity of Lectin from Seeds of Pongamia Glabra

S. Sheela Devi*, J. Dhanalakshmi, S. Selvi

Department of Biochemistry, Bharathidasan College of Arts and Science, Erode-638116, Tamil Nadu, India

*Corresponding author email id: sheel20bio@gmail.com

 

Abstract

Lectins are non immunogenic proteins that selectively bind carbohydrates without processing them enzymatically. They have wide range of applications in agricultural and biomedical research and it is useful in the antidotal treatment of snake bite. Seed lectins can be attributed to the importance of legume seeds as a rich source of dietary protein. The Pongamia glabra lectins were prepared, molecular characterization was done and the anti-fungal and antibacterial activity were determined. The total carbohydrate was estimated with DNS method and protein by CBB method. The lectin isolated from Pongamia glabra seed is a most stable and it shows the high activity at pH 5-9 and the temperature at 60°C. It is concluded that Pongamia glabra exhibit antifungal and bacterial activity.  It is widely used in agricultural and biomedical research and it also act as an insecticide.

 

Key words:

Pongamia glabra, Lectin, Candida albicans, antifungal activity, antibacterial activity.

 

Introduction

Lectins are non-immunogenic proteins or glycoproteins that selectively bind carbohydrates without processing them enzymatically. They are widespread in the biosphere and occur in essentially every living organism, the richest source being the storage organs of higher plants. Compositions, structures and biological functions have been reviewed repeatedly. In the Plant Kingdom, seeds of legumes (Leguminosae) have long been known to be a rich source of lectins. Legume lectins are the best studied group of plant lectins and hundreds of these proteins have been isolated and characterized in relation to their chemical, physicochemical, structural and biological properties. Increasing experimental evidence suggest that seed lectins in legumes are defense proteins that may protect mature seeds against the attack of predators such as insects and mammals. Indeed, many legume lectins exhibiting different carbohydrate specificities are insecticidal to important pests of crops. This biological activity of plant lectins is of great economic potential because lectin genes are good candidates to confer insect resistance to transgenic crops. Because of their ability to specially recognize unique carbohydrate structures, lectins are being used in the purification and characterization of glycoconjugates and in the study of cell-surface architecture. Lectins are a large and heterogeneous group of proteins that have the ability to bind reversibly to mono and oligosaccharides (Van damme et al., 1998). Although lectins have been identified and characterized from several plant species, taxa and organ types, the largest contribution to our understanding of the molecular and biochemical properties of plant lectins has come from studies of legume seed lectins (Sharon et al., 1990). seed lectins can also be attributed to the importance of legume seeds as a rich source of dietary protein, additionally, lectins are also found to be useful in the fractionation of cells for their use in bone marrow transplantation, hence the need to study their protein composition.  

Although typical lectin  levels  in  legume  seeds  range from  0.1 ±5%  of  total seed protein, values of up to 50% have been reported in some species such as those in the genus Phaseolus. At present, detailed knowledge about the molecular, bio- chemical and physicochemical properties of several lectins, their carbohydrate binding, including sugar, specificities and their applications in agricultural and biomedical research is available (Van damme et al., 1998). Pongamia glabra synonyms Pongamia pinnata (Papilionaceae) is a tree, commonly known as Karanj in India. Leaves are alternate, imparipinnate. Flowers are in lax auxiliary racemes. Seeds are reniform and thick. Helium of seed is small. Root and bark are hot, acrid and used as antihelminthic as well as useful in the diseases of eye and skin, good for tumours, piles, wounds, ulcers, itching and urinary discharges. Leaves are digestive, laxative, antihelminthic and useful in wounds and inflammation. Flowers are used as antidiabetic. Fruits and seeds are used as antihelminthic and useful in keratitis and urinary discharges. Oil is also used as antihelminthic and useful in rheumatism, leucoderma, scabies, wounds, leprosy, piles and ulcers. Every part of the plant is useful in the antidotal treatment of snakebite (Kirtikar et al., 1987). In screening for potent antifungal activity of lectin from various plant seed, we found that the extract from Karanj seed exhibits strong inhibition of growth of the Candida albicans   pathogens tested. This project represented lectin isolation, purification, characterization, anti-fungal activity and antibacterial activity from the seed extract of Pongamia glabra.

 

Materials and Methods

 

     1) Pongamia glabra plant                             2) Pongamia glabra seeds

          

                         Fig.1                                                        Fig.2

 

Pongamia glabra seeds were obtained from Gloris Biomed Research Centre Herbal garden, Villupuram, Tamilnadu, India.

 

Lectin extraction:

About 100 g of deshelled Pongamia glabra seeds were homogenized in a kitchen blender and defatted with 3x300 ml of distilled acetone. The defatted seed-meal was dried, mixed with 1 of 20 mM phosphate buffer, pH 7.4, containing 0.15 M NaCl and 10 mM β-mercaptoethanol (PBS- β ME), and stirred at 48°C for 12 hrs. This suspension was then filtered and the filtrate centrifuged at 6000 rpm for 30 min at 4°C in a Remi C23 refrigerated centrifuge. The supernatant was subjected to ammonium sulphate precipitation.

 

Culture collection and maintenance:

The organisms were obtained from Madras Medical College Chennai and confirmed by Germ-tube test, production of chlamydoconidia on Corn meal agar and growth at 45°C. Isolates were maintained on Sabouraud’s Dextrose Agar (SDA) at 4°C. Organisms were sub cultured on SDA and incubated at 37°C for 24 h. Several colonies of each Candida species were collected in 2 ml of sterile Phosphate Buffered Saline (PBS) to prepare a suspension. The suspension was adjusted to 70% Transmittance (T) by a spectrophotometer at 530 nm. This should result in a suspension containing about 1X106 cfu per ml.

 

Analytical methods:

Carbohydrate content:

The neutral sugar was estimated by the phenol-sulfuric acid method of using glucose as the standard (Dubois et al., 1956).

 

Protein concentration:

This was determined by the Bradford method (Bradford, 1976) using Bovine Serum Albumin (BSA) as standard. Readings at 280nm were also used to determine protein content of the column eluates.

 

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE):

The polyacrilamide gel electrophoresis in Sodium Dodecyl Sulfate (SDS) for purified lectin was performed in vertical 1 mm slab gels, according to the method of Laemmli (1970) using 4% and 17.6% stacking and resolving gels, respectively. Samples (lectins and the molecular weight markers) were dissolved in 0.01M sodium phosphate pH 7.0, 2% SDS buffer with 1% β-mercaptoethanol and incubated at 100oC for 15 min. A few crystals of sucrose were dissolved in the samples which were then applied to the gel. The electrophoresis was conducted at a constant current of 13 mA for 4 hrs. The protein bands were visualized by staining with Coomassie Brilliant Blue R-250. Estimation of apparent molecular mass of the lectin subunit and fragments were made using a mixture of proteins (phosphorilase B, 94.4 kDa, carbonic anhydrase, 29.0 kDa, myoglobin, 17.5 kDa and Lysozyme.

 

Antifungal activity of lectin:

Antifungal activity was performed on sterile Petri plates (100x15 mm) containing 10 ml Dox’s agar sterilized at 15 psi and 120°C for 20 min. Sterile paper disks, 1 cm in diameter, were placed at the surface of heavily seeded medium with the tested organism. A 10 μl aliquot of the purified lectin in 0.05 M Sodium Phosphate buffer pH 7.0 was added to the disk. Petri dish was incubated at 37°C for 48 hrs, at the end of which the diameter of the clear zone of inhibition surrounding the sample was taken as a measure of the inhibitory power of the sample against the particular test organism (Irob et al., 1996).

 

Antibacterial   activity   of lectin:

Antibacterial activity of Pongamia glabra seed lectin was investigated by the disc diffusion method (Cole et al., 1994). The microbial strains were obtained from stock cultures in nutrient agar (0.7%). One-hundred millilitres of warm Nutrient Agar (NA) (43°C) and 0.5 ml of bacteria suspension (105-106 CFU /m1) were mixed and 10 ml volumes were distributed in sterile Petri plates (90 X 15 mm) and allowed to solidify. Sterile blank paper discs (6 mm diameter) impregnated with 20µl of sterile solution of purified lectin of Pongamia glabra (1.0mg/ml, 2.0 mg /ml), and positive control (amoxicillin, 2.5 mg /ml) were added on the agar plates. Plates were incubated at 37°C for 24 hrs.  A transparent ring around the paper disc revealed antimicrobial activity. Zones of growth inhibition around discs were measured in millimeters.

 

Determination of the Minimum Inhibitory Concentration and the Minimum Bactericidal Concentration:

Minimum Inhibitory Concentration corresponded to the minimum lectin concentration that inhibited visible bacterial growth. MIC was determined by the dilution tube test (Courvalin et al., 1985). Serial dilutions of purified lectin of Pongamia glabra seeds   in PBS were prepared and added to the bacteria cultures in Nutrient Broth (NB) containing 107 cells ml-1 in the exponential growth phase (turbidity equivalent to 0.5 in the McFarland scale). The samples were incubated for 24 hrs at room temperature (25°C). Afterwards, cultures were seeded onto NA and incubated for 24 hrs at room temperature (25°C). The Minimum Bactericidal Concentration (MBC) corresponds to the minimum concentration of the lectin that reduced the number of CFU to 0.1% of the initial concentration.

 

 

Measurement of zone of inhibition:

The zone of inhibition for each sample was observed, measured and expressed in mm. From this the activity index (A.I.) and Percent Inhibition (P.I.) were calculated for all lectin and extracts using the following formula (Lis et al., 1998). Refer table 2.

A.I = Mean zone of inhibition of each solvent extract

 Zone of inhibition obtained for standard

 P.I. = Activity index x 100

 

Results

Purification of the Lectin from Pongamia glabra seeds:                                             

Pongamia glabra lectin was purified by extraction of the seed meal in 0.15 M Nacl followed by affinity chromatography on sephadex G-50 column as showed. The lectin was retained by the Sephadex matrix and was eluted (peak iii) after application of 0.1 M glucose in the equilibrium buffer. The antifungal lectin of Pongamia glabra was purified to homogeneity according to the procedure summarized in Table-1.

Table-1: Purification of the Lectin from Pongamia glabra seeds

Purification stage

Volume/ ml

Protein mg/ml

Total protein mg/ml

Total lectin (titerbXml)

Specific c activity (HU/mg)

Purification foldd

Yield(%)e

Alcolic crude extracta

100

40

4000

12800

3.20

1

100

Dialysis

50

25

1250

6400

5.12

1.6

50

Sephadex G-100

30

8

240

2840

16

5

30

 

a Crude protein extract from 10 g of cotyledons.

b Minimal concentration of protein able to cause visible agglutination of a 2–4% suspension of rabbit erythrocytes.

c Specific activity is defined as the hemagglutination unit (HU) divided by the protein concentration (mg/ml) of the assay solution. Rabbit erythrocyteswere used for the assay.

d Purification index was calculated as the ratio between the minimal concentration of the crude extract able to cause visible agglutination of the rabbit erythrocytes and that of the protein fraction obtained at each purification step.

e The lectin was obtained by affinity chromatography on Sephadex G-100.

 

Fig 3: Purification of the lectin from Pongamia glabra by column chromatography on Sephadex G-50 column.

 

Molecular Mass determination of purified antifungal protein by SDS-PAGE:

The purified protein (3.0 μg) was analyzed on SDS-PAGE (10% w/v) and stained with silver nitrate. On Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis the purified antifungal protein showed a single band indicating that it was electrophoretically homogeneous. The molecular mass of the purified antifungal lectin was determined as 20 kDa by comparing with relative mobility of the molecular mass of protein ladder (Fig 4).

Fig 4: Molecular Mass determination of purified antifungal protein by SDS-PAGE

 

Antifungal activity of purified lectin from seeds of Pongaia glabra on Candida albicans:

Growth inhibition of Candida albicans was noticed in the presence of Pongamia glabra lectin at several serial dilutions (Fig-5) where the original concentration was (100 mg/ml). Purified lectin inhibited the growth of Candida albicans with LIC of 0.2, 0.02, 0.1 and 0.01 mg/ml, respectively and the antifungal activity compared with standard antibiotic.

Fig 5: Antifungal activity of purified lectin from seeds of Pongamia glabra on Candida albicans

 

 

Antibacterial activity of lectin:

Table 2: Diameter of zone of inhibition shown by Pongamia glabra lectin against standard antibiotic.

S.No

Organism

Clearing zone in diameter(mm)

MIC(µg/ml)

MBC(µg/ml)

Antibiotics

1

Staphylococcus aureus (+)

20.0

1.5

16.5

amoxicillin

2

Bacillus subtilis (+)

12.0

16.5

180.0

amoxicillin

3

Pseudomonas aeruginosa(-)

18.6

1.5

16.5

amoxicillin

 

Fig 6: Lectin against Staphylococcus aureus  

 

Fig 7: Lectin against Bacillus subtilis 

 

Fig 8:   Lectin against  Pseudomonas aeruginosa

 

Discussion

The aim of the present study was to isolate a lectin from Pongamia glabra seeds and investigate its antifungal activity. The lectin could be successfully purified in a single step by affinity chromatography on Sephadex G-100. Loading of pre-purified extract on an affinity column followed by washing out the unbound proteins then eluting the bound lectin with 250 mM glucose led to increments in the specific activity up to 320 titer/mg corresponding to 80% yield. The final obtained lectin yield was ca. 160 mg per 100 g dry seed weight. This value is exactly the same as obtained by Rudiger et al. (1993). SDS-electrophoretic patterns of purified Pongaia glabra lectin in the presence and absence of  β-mercaptoethanol manifested two bands of 5.54 and 19.3 kDa corresponding to α and β chains, non-covalently bound. Hence, the bands seen in SDS–PAGE most likely represented the subunits of a dimeric lectin; a common occurrence in plant lectins (Van damme et al., 1998). The observed antifungal activity of Pongamia glabra against Candida albicans agrees with the results obtained from other plant legume lectins (Yan et al., 2005; Ye et al., 2001). This activity was concluded to be related to the lectin carbohydrate binding property (Damico et al., 2003) that might endow lectin molecules with binding activity towards certain carbohydrate components in the fungal cell wall affecting its activity and viability as most lectins recognize either N-acetylneuraminic acid, N-acetylglucosamine, N-acetylgalactosamine, galactose, mannose, or fucose in accordance with the previous conclusion (Lis et al., 1998). The studied lectin showed a remarkable antibacterial activity against all the tested bacteria, while an irrelevant antibacterial activity was detected with the Pongamia glabra seed lectin showed higher antibacterial activity than the other parts of the plant and have an important role in the protection of seeds against microbial invaders. These results demonstrate that pongamia seed lectin is responsible for the antibacterial action observed in the seed extract. An outstanding feature of the antibacterial activity of the isolated lectin is its nonselective activity against bacteria and its activity on several species of human pathogenic bacteria.

 

Conclusion

The lectin isolated and purified from Pongamia glabra seed is a most stable and it shows high activity at pH 5-9 and the temperature at 60°C. Concluded that Pongamia glabra lectin is very potent in haemagglutination and antifungal activity. And its widely used in agricultural and biomedical research, and it also acts as an insecticide. The use of advanced statistical designs has helped to identify the optimum conditions. It has been concluded from the results of this investigation that the Pongamia glabra seed lectin are good inhibitors of bacterial and fungal growth.

 

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