Antioxidant and antimicrobial activities of Penicillium sp. lectins

Lectins are a diverse group of proteins of non-immune origin that interact specifically with glycans. Owing to their specificity, they can mediate various cellular and molecular recognition processes. To explore information on biological activities of lectins from Penicillium duclauxii, P. proteolyticum and P. griseoroseum, they were investigated for their antioxidant and antimicrobial activities. Penicillium sp. lectins exhibited moderate antioxidant activity. P. duclauxii, P. proteolyticum and P. griseoroseum lectins inhibited DPPH with an IC50 value of 71.42, 75.04 and 82.11 μg/mL, respectively. P. duclauxii, P. proteolyticum and P. griseoroseum lectins inhibited the hydrogen peroxide radical with IC50 values of 198.57, 209.76 and 215.31 μg/mL, respectively. P. duclauxii and P. proteolyticum lectins exhibited potent antibacterial activity against Gram-negative bacteria. P. griseoroseum lectin inhibited only Gram-positive bacteria. Penicillium sp. lectins did not exhibit antifungal activity. The biological potential of Penicillium sp. lectins will help to understand their biomedical applications. This is the first report on the antioxidant and antimicrobial activities of purified lectins from Penicillium sp.

Antioxidants are molecules that donate electrons/ hydrogen to radicals and quench free radical reac-tions based on their reduction potential or other traits.During normal or pathological cell metabolism, free radicals are generated that have one or more unpaired electrons (superoxide, hydroxyl, peroxyl), and the compounds that have the potential to scavenge free radicals can play a role in curing diseased cells and improving pathological conditions.Thus, they play a significant role in defense mechanisms of organisms against various pathologies related to free radical attack [32].There has been an increase in research into new compounds with antioxidant activity in natural products.Many plant lectins have been reported to possess an antioxidant potential [23,27,28].However, there is only one report on the antioxidant potential of lectins from microfungi [33].
Lectins from various sources have also been explored for their potential antimicrobial activity [34].The carbohydrate-binding sites of lectins can interact with carbohydrates present on the surface of microorganisms and exert a antimicrobial effect.Thus, different carbohydrate specificities of lectins greatly influence recognized targets [35].There are only a few reports on the antimicrobial action of lectins from microfungi.The antimicrobial activities of lectins from Fusarium sp.[36], Aspergillus sp.[37,38] and Penicillium sp.[39] have been reported.New lectins have been reported from Penicillium sp.[11,12,39], however, scant information is available in the literature regarding their biological potential.In previous studies, lectins from Penicillium sp. were purified, characterized and evaluated for their mitogenicity [40,41].The aim of the current study was to explore the antioxidative and antimicrobial activities of these purified Penicillium sp.lectins.

Ethical statement
Animal care was taken according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) of the Government of India.Experimental work was approved by the Institutional Ethics Committee for Animals (Permit No. 107/99/CPCSEA/201429).

Extraction and purification of Penicillium sp. lectins
Fungal cultures of P. duclauxii MTCC 7997, P. proteolyticum MTCC 10300 and P. griseoroseum MTCC 9624 were obtained from MTCC housed at IMTECH, Chandigarh, India.All the cultures were maintained and cultivated on their respective medium under growth conditions specified by MTCC, as described earlier [12].Fungal mycelia were harvested by filtration from broth cultures and intracellular Penicillium sp.lectins were extracted in 0.1 M phosphate buffered saline (PBS), pH 7.2.as described [14].Penicillium sp.lectins were purified by ion chromatography on a DEAE Sepharose column (GE Healthcare, USA) and gel exclusion chromatography on a Sephadex G-100 column following the reported methodology [40,41].

Hemagglutination assay and protein quantification
Rabbit erythrocytes were used for the hemagglutination assay with Penicillium lectins as described [14].Hemagglutination titer was used to express lectin activity, which is defined as the reciprocal of the highest dilution that exhibits visible erythrocyte hemagglutination.Protein quantification of Penicillium sp.lectins was performed by the Lowry method [42].Purified lectins were then evaluated for their antioxidant potential and antimicrobial activity.

DPPH (2,2-diphenyl-1picrylhydrazyl) radical scavenging assay
The antioxidant activity of Penicillium sp.lectins was determined by DPPH radical scavenging [43].The radical scavenging effect of lectins was studied using DPPH, a stable free radical.The DPPH-based photometric measurement is widely used for antioxidant screening as it is a simple and rapid method.A purified Penicillium lectin solution (0.1 mL) in phosphate buffer at different concentrations (1,5,10,25,50,75, 100 µg/mL) was added to a methanolic solution of DPPH (3.9 mL).The reaction mixture (4 mL) was shaken vigorously and then incubated for 30 min in the dark at room temperature.Absorbance of the resultant solution was read spectrophotometrically at 517 nm against the blank (methanol plus lectin solution).The control solution contained all reagents except the test sample.Owing to the strong reducing power and weak metal chelating ability, ascorbic acid at different concentrations (1,5,10,25,50,75, 100 µg/ mL) served as a standard.The DPPH radical scavenging activity of lectins was determined by converting the absorbance into the percentage antioxidant activity using the following equation:

Percent inhibition (%) =
where Abs A is the absorbance of the control reaction and Abs B is the absorbance of the lectin solution.
The concentration of the test sample required to inhibit 50% of the DPPH free radical is defined by the IC 50 (half maximal inhibitory concentration) value.Higher free radical (antioxidant) activity is indicated by lower IC 50 values of the reaction mixture.

Hydrogen peroxide radical scavenging assay
The ability of Penicillium sp. lectin solution to scavenge hydrogen peroxide radicals was determined as described [44].where Abs A is the absorbance of the control reaction and Abs B is the absorbance of the lectin solution.The half maximal inhibitory concentration (IC 50 ) was determined as described above.

Antimicrobial assay
The antimicrobial activity of purified Penicillium sp.lectins was assessed against various bacterial and fungal cultures by the disk diffusion method [45].

Antibacterial assay
The bacterial cell suspension was prepared by inoculating the test culture strains from nutrient agar slants into nutrient broth medium and grown for 24 h by shaking (150 rpm) at 37°C.The overnight-grown bacterial cultures were adjusted with sterile PBS (pH 7.4) to yield a 0.5 McFarland suspension.The prepared bacterial inoculum (100 µL) was then spread on Muller-Hinton agar plates by the three-dimension swab technique.Sterile filter paper disks (6.0 mm diameter, Whatman, U.K.) were placed on the inoculated agar plates and each disk was impregnated with purified Penicillium sp. lectin (50 µg).Inoculated agar plates were then incubated at 37°C for 24 h.Reference antibiotics, ciprofloxacin (10 µg) and gentamicin (50 µg), served as a positive control.The degree of susceptibility of the test organism was determined by examining the zone of inhibition around the sample disk.Bacterial growth encircled around the disks indicated the non-inhibitory action of lectin, whereas crescents of inhibition around disks depicted antibacterial activity.

Antifungal assay
Yeast cultures were grown in YEPD broth for 48 h at 30°C.Fungal cultures of A. flavus and F. oxysporum were grown on CYA and PDA, respectively, for 5 days at 25°C.The fungal suspensions (10 7 spores/mL) and yeast cells (10 7 cells/mL) were prepared, and spreading was performed on 2% (w/v) malt extract agar (MEA) plates.Purified Penicillium sp.lectins (50 µg) were applied on sterile filter paper disks as described above.Nystatin (100 units) and amphotericin B (50 µg) were used as the positive control.The zone of inhibition around the sample disk was detected after incubation for 72 h at 30°C for determination of the inhibitory potential.

Minimum inhibitory concentration (MIC) determination for antibacterial activity
Purified Penicillium sp.lectins were serially diluted with sterile water to different concentrations (5-50 µg) for the determination of MIC.Bacterial cultures (100 µL) were spread over Muller-Hinton agar plates.Different lectin concentrations were impregnated over sterile filter paper disks placed on agar plates.The disk diffusion method was performed to determine the inhibition of microorganisms.The MIC was taken to be the lowest concentration that inhibited bacterial growth.

Statistical analysis
All the experiments were repeated three times and the results are expressed as the mean±standard deviation of triplicate values.

RESULTS
Lectins from P. duclauxii, P. proteolyticum and P. griseoroseum were purified to the following specific activities: 131.1, 168.42 and 124.27 titer/mg, respectively, as described previously [40,41].All lectins were used for determination of the antioxidant potential, and antifungal and antibacterial activities.

DPPH free radical scavenging by Penicillium sp. lectins
To evaluate the antioxidant activity, the DPPH free radical scavenging assay is considered the most accurate screening method.The percentage inhibition of DPPH by purified Penicillium sp.lectins and ascorbic acid (standard) was determined (Table 1).Penicillium sp.lectins possess a moderate antioxidant capacity.The highest scavenging action was shown by P. duclauxii lectin.IC 50 values of 71.42 µg/mL, 75.04 µg/mL and 82.14 µg/mL were shown by P. duclauxii, P. proteolyticum and P. griseoroseum lectins, respectively, whereas ascorbic acid had an IC 50 value of 48.80 µg/mL.

Hydrogen peroxide scavenging by Penicillium sp. lectins
The percentage inhibition of hydrogen peroxide by purified Penicillium sp.lectins and the standard, ascorbic acid, were also determined (Table 2).The IC 50 values of P. duclauxii, P. proteolyticum and P. griseoroseum lectins for hydrogen peroxide were found at concentrations of 198.57µg/mL, 209.76 µg/mL and 215.31 µg/mL, respectively.Ascorbic acid was used as the standard as it is a very strong antioxidant, and 50% scavenging of hydrogen peroxide radicals was achieved at a concentration of 150 µg/mL (IC 50 ).

Antimicrobial activity of Penicillium sp. lectins
The purified Penicillium sp.lectins were screened for antimicrobial activities by the disk diffusion method.The zone of inhibition by Penicillium sp.lectins was determined by measuring the diameters of the inhibitory zones (Table 3).P. duclauxii lectin exhibited an enhanced inhibitory effect over Gram-negative bacteria as compared to Gram-positive bacteria.A high antibacterial activity of P. duclauxii lectin was observed against E. coli and P. aeruginosa, with maximum zones of inhibition of 38±0.07 mm and 35±0.13 mm, respectively.P. proteolyticum lectin showed inhibitory zones of 27±0.88 mm and 22±0.19 mm against E. coli and P. aeruginosa, respectively.P. duclauxii lectin expressed low antibacterial activity against S. aureus (15±0.24mm), whereas P. proteolyticum lectin was non-inhibitory against it.Growth of B. cereus remained unaffected with both lectins.However, P. griseoroseum lectin strongly inhibited Gram-positive bacteria, including S. aureus (27±0.39)and B. cereus (28±.43),but was non-inhibitory towards Gram-negative bacteria.
The growth of yeast (C.albicans, S. cerevisiae) and fungi (A.niger, F. oxysporum) was not affected in the assay.The MIC of purified Penicillium sp.lectins against bacterial strains was determined (Table 4).In the case of P. duclauxii lectin, the MIC was lowest for E. coli (7.5 µg) and highest for S. aureus (45 µg), whereas, P griseoroseum lectin had an MIC of 17.5 µg towards B. cereus.

DISCUSSION
Oxidation processes associated with aerobic life forms can lead to cell and tissue damage as the result of the generation of increased amounts of reactive oxygen species (ROS) [46].Antioxidants delay or inhibit this oxidation process and thus play a significant role in the organism's defense against pathologies related to high ROS levels [32].DPPH is a widely used assay for determining antioxidant activity [47].P. duclauxii, P. proteolyticum and P. griseoroseum lectins exhibited concentration-dependent antioxidant activities.Interaction between lectin molecule (antioxidant) and DPPH radical can lead to a decrease in absorbance of DPPH radicals owing to scavenging of the radical by hydrogen donation [33].Penicillium sp.extract was reported to express a DPPH free radical scavenging activity of about 50% at a concentration of 300 µg/mL [48].Concentration-dependent free radical scavenging of DPPH is expressed by some endophytic fungal lectins [33].
P. duclauxii, P. proteolyticum and P. griseoroseum lectins exhibit antioxidant activities.The highest hydrogen peroxide scavenging activity was shown by P. duclauxii lectin.Hydrogen peroxide is a nonradical derivative of oxygen and can inactivate some enzymes.It is a weak oxidizing agent and can oxidize essential thiol (-SH) groups.It can react with different molecules in living organism after entry into cells [49].A concentration-dependent hydrogen peroxide scavenging activity was also displayed by lectins from endophytic fungi [33].Cratylia mollis (pCramoll) and recombinant Cramoll 1 (rCramoll 1) lectins exhibited concentration-dependent attenuation of H 2 O 2induced oxidative stress [49].Thus, antioxidants have gained interest owing to their role in preventing oxidative-stress related diseases caused by free radical attack on key biological components, such as nucleic acids and lipids [30].
Microorganisms usually possess carbohydrates on their cell surface that are either covalently bound (glycosylated teichoic acids to peptidoglycan) or noncovalently bound (capsular polysaccharides) and act as potential lectin-reactive site [35].Owing to the ability of lectins to form complexes with microbial glycoconjugates, they can play a key role in the recognition of microbes and thus exert antimicrobial activity.

P. duclauxii and P. proteolyticum lectins inhibited
Gram-negative bacteria, whereas P. griseoroseum lectin inhibited Gram-positive bacteria.P. proteo- lyticum and P. griseoroseum lectins exhibited strong selective antibacterial activity.Interactions of lectin with lipopolysaccharides present in the cell wall of Gram-negative bacteria could be responsible for its antibacterial action [50].Lectins could also interact with N-acetyl-D-glucosamine, N-acetylmuramic acid and tetrapeptides linked to N-acetylmuramic acid and present in the cell wall of Gram-positive bacteria [50].P. duclauxii [40] and P. proteolyticum [41] lectins are nonspecific towards N-acetyl-D-glucosamine.Thus, their non-inhibitory action towards Gram-positive bacteria (which possess high levels of N-acetyl-Dglucosamine in their cell wall) further corroborates this.However, P. griseoroseum lectin exhibits specificity towards N-acetyl-D-glucosamine [12] and inhibits Gram-positive bacteria, which substantiates lectinglycan interaction leading to bacterial inhibition.In the case of S. aureus, P. duclauxii lectin might form a channel on the cell membrane resulting in an outflow of cellular contents, thus leading to cell death.The thicker murein layer in the case of Bacillus cereus probably acts as a barrier in P. duclauxii and P. proteolyticum lectins and thus does not exhibit any inhibitory action.
Owing to the potential antioxidant and antibacterial actions of Penicillium sp.lectins, these compounds could have prospective biomedical applications and may be used further for microbiology studies.
Funding: Punjabi University, Patiala, India is acknowledged for funding the experiments.

Table 1 .
DPPH radical scavenging by the standard (ascorbic acid) and purified Penicillium sp.lectins.

Table 2 .
Hydrogen peroxide radical scavenging by the standard (ascorbic acid) and purified Penicillium sp.lectins.
[12]can specificity of lectins from Penicillium sp.[12]NA:Not applicable NS: Zone of inhibition not shown by lectins