In vitro Antifungal, Antioxidant and Cytotoxic Activities of a Partially Purified Protein Fraction from Atlantia monophylla Linn (Rutaceae) Leaf

Purpose: To determine the in vitro antifungal and antioxidant activities of the aqueous extract and protein fraction of Atlantia monophylla Linn (Rutaceae) leaf. Methods: Ammonium sulphate (0 – 80 %) precipitation method was used to extract protein from the leaves of A. monophylla Linn (Rutaceae). In vitro antifungal assays were performed by disc-diffusion and micro-broth dilution methods. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), hydrogen peroxide (H 2 O 2 ), superoxide and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activities were performed to evaluate in vitro antioxidant activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was employed to assess the molecular weight of the protein fractions. Protein concentration was determined by Bradford method. The cytotoxicity of these extracts was tested on Vero cell lines. Results: Both the aqueous extract and protein fraction (AMP III) of Atlantia monophylla leaf exhibited higher antifungal activity on Candida albicans than on Aspergillus fumigatus. AMP III fraction showed greater in vitro antioxidant activity than the aqueous extract. SDS-PAGE analyses revealed the presence of two protein bands with molecular weight approximately of 16 and 67 KDa in AMP III. Protein concentration was 240 µg/ml in the aqueous extract and 670 µg/ml in AMP III fraction. The aqueous extract and protein fraction exhibited cytotoxicity at concentrations > 100 µg/ml on Vero cells. Conclusion: Plant-derived proteins/peptides possessing antifungal and antioxidant properties would be a good alternative preparation for the treatment of infectious diseases.


INTRODUCTION
Fungal infections remain a significant cause of morbidity and mortality despite advances in medicine and the emergence of new antifungal agents. Increasing incidents of fungal resistance following opportunistic pathogen infections such as Human Immunodeficiency Virus (HIV), patients with immunosuppressive chemotherapy led researchers to look for new, safer and more effective antifungal agents as an alternative strategy to the available drugs in the market [1]. Due to these pathological conditions, there are alterations in normal metabolism that may generate reactive oxygen species and other free radicals in the body. Reactive oxygen species (ROS) is a collective term that encompasses radical and non-radical derivatives of molecular oxygen that inflict injuries to the tissues through lipid peroxidation resulting in atherosclerosis, cancer, diabetes and other diseases [2]. The schematic search of higher plants for antifungal activity has shown that some plants extracts may have the ability to retard fungal growth or completely inhibit the fungus.
Many low molecular mass proteins or peptides have been reported to possess antifungal and antioxidant activities. Huynh et al [3] isolated low molecular mass proteins which are involved in a defense mechanism against fungi. Chen et al [4] studied the antioxidant activities of synthetic peptides based on an antioxidant peptide isolated from the soy protein hydrolizates.
Medicinal plants have been the basis for the treatment of various infectious diseases in traditional medicine, and possess a variety of compounds of known therapeutic properties. Much attention has been paid to plant-derived antifungal and antioxidant compounds, based on the knowledge that plants have their own defense system [5]. Only a small fraction of the known plant species of the world have been evaluated for the presence of antimicrobial and antioxidant compounds, and thus it is necessary to increase the efforts in collecting and screening for the development of novel and environmentally safe therapeutic agents.
Atlantia monophylla Linn (A. monophylla) belongs to the family of Rutaceae commonly called as Jungli Nimbu. The main objective of the present study was to determine the in vitro antifungal and antioxidant activities of a partially purified protein from the leaves of A. monophylla Linn (Rutaceae).

Fungal strains
Candida albicans 10231 and Aspergillus fumigatus 1022 used in the present study are of American Type Cell Culture (ATCC) procured from IMTECH, Chandigarh, India.

Plant material
The leaves of Atlantia monophylla Linn (Rutaceae) were collected in the month of October 2008, from Tirumala hills, Chittoor District, Andhra Pradesh, India. The plant was authenticated by Prof N Yasadomma, Department of Botany, Sri Venkateswara University, India. A voucher specimen was deposited in the herbarium as SVU/B/AM-32.

Protein extraction from aqueous extract
Leaf powder (100 g) of A. monophylla Linn (Rutaceae) was placed in a corning jar; 300 ml of water was added and soaked for 48 h. Protein fractions were isolated from the aqueous extract using ammonium sulphate precipitation from 0-20, 20-40, 40-60 and 60-80 % saturation, followed by centrifugation at 10000 x g for 10 min at 4 °C. The supernatants were dialyzed with 10 mM phosphate buffer, pH 7.4 and the resultant protein fractions designated as AMP I, AMP II, AMP III, and AMP IV were subjected to antifungal, antioxidant and cytotoxicity studies.

Protein concentration determination
The protein contents of the aqueous extract and dialyzed samples of the leaves were determined with Bradford method [6] using bovine serum albumin (BSA) as standard.

Disc-diffusion method
Antifungal activity of the aqueous extract of A. monophylla Linn (Rutacea) fractionated with ammonium sulphate precipitation was tested using the diffusion method according to Bauer et al [7]. The fungal strains were maintained on potato dextrose agar (PDA) medium (Hi-Media). Sterile discs of Whatmann No.1 filter paper of about 6 mm diameter were impregnated on the surface with the media. Different concentrations of both aqueous and protein fractions were prepared and applied on the discs and incubated for 48-72 h at 28 °C. The results were recorded by measuring the inhibitory zone around the discs.

Micro-broth dilution method
Evaluation of the susceptibility of Candida albicans and Aspergillus fumigatus species were performed using the micro broth dilution method according to M27-A2 for yeast guidelines [8].
Yeast strains were grown aerobically overnight at 35 °C on Sabouraud dextrose agar plates.

Electrophoresis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli and Favre [9]. It was carried out on Bio-Rad gels composed of stacking gel (5 % w/v) using 1.0 M Tris-glycine buffer containing 0.4 % SDS at pH 6.8 and resolving gel (12 % w/v) using 1.5 M Tris-glycine buffer containing 0.4 % SDS at pH 8.8. Protein sample was dissolved in phosphate buffer (5 mg/ml) and mixed with a solubilization buffer Tris -HCl 6.22 mμ (pH 6.8) which contains 2 % w/v) SDS, 50 % glycerol, a pinch of bromophenol blue and reduced with 0.9 mμ 2-mercaptoethanol in boiling water for 3 min. Protein sample was loaded onto each well and electrophoresis (Bio-Rad electrophoresis apparatus, Bio-Rad Laboratories, Hercules, CA) was conducted at constant current of 60 volts by a Bio-Rad electrophoresis constant power supply unit (Model 200/2, Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA). After electrophoresis, gels were stained with CBB. Molecular masses were determined using the molecular weight standard kit from GENEI, Bangalore, India.

DPPH free radical scavenging activity
Free radical scavenging activity was determined using the 2, 2′-diphenly-1-picrylhydrazyl (DPPH) method of Burits and Bucar [10]. One ml of various concentrations of the extract in methanol was added to 4 ml of 0.004 % methanol solution of DPPH. After a 30-min incubation period at room temperature, the absorbance was read against a blank at 517 nm. The inhibition of free radicals by the extract in percentage terms (I %) was calculated using equation 1.
I % = {(Ac -As)/Ac} × 100 …………………… (1) where Ac is the absorbance of the control reaction (containing all reagents except the test compound), and As is the absorbance of the reaction mixture containing the test compound.

Superoxide anion scavenging activity
Measurement of superoxide anion scavenging activity was carried out according to Gulcin et al. [11]. Tris-HCl buffer (1.5 ml, pH 8.0, 32 mM), 0.5 ml of nitroblue tetrazolium (NBT, 300 μM), 0.5 ml of reduced nicotinamide adenine dinucleotide (NADH) solution of 468 μM and different concentrations of extract (10 -250 μg/ml) were taken. The reaction was initiated by the addition of 1 mL phenozine methosulphate (PMS) (30 μM) to the mixture. The reaction mixture was incubated at 25 °C for 5 min and absorbance was measured at 560 nm. Decreased absorbance of the reaction mixture indicates the increased superoxide anion scavenging activity. Ascorbic acid was used as a standard and inhibition calculated.

ABTS radical scavenging activity
The antioxidant activity was determined with stable 2, 2'-azinobis-(3-ethylbenzothiazoline-6sulfonic acid (ABTS+) cation radical method of Re et al [13]. ABTS (2 mM) was prepared by dissolving in 50 ml of phosphate buffered saline (pH 7.4). ABTS+ was produced by reacting 50 ml of stock solution with 200 μl of 70 mM potassium persulfate (K 2 S 2 O 8 ) water solution. The mixture was left to stand in the dark at room temperature for 15-16 h before use. For the evaluation of antioxidant activity, the ABTS+ solution was diluted with PBS to obtain the absorbency of 0.8 ± 0.03 at 734 nm. Different concentrations of both the extracts were prepared and mixed with 3 ml of ABTS solution. The absorbance was read at room temperature after 10 min at 734 nm. PBS solution was used as a blank sample. The percentage of inhibition was calculated.

Cytotoxicity assay
Vero cell lines were maintained in RPMI 1640 medium supplemented with 10 % FBS, 1 mM sodium pyruvate and were grown in an incubator at 37 ºC, 5 % CO 2 in a humidified atmosphere. Aqueous extract and protein fraction (AMP III) were dissolved in water and its final concentration was made to 2 % in each well. The confluent cell culture was trypsinized and the cell count was adjusted to 5.4 x 10 5 cells/ml. To each well of the 96-well microtiter plate, 0.1 ml of the diluted cell suspension (25000 cells/well) was added and kept for 24 h in an incubator at 37 ºC in 5 % CO 2 atmosphere for cell monolayer formation. After 24 h, the media was flicked off, washed and 100 µl of different concentrations of the compounds were added to the cells in microtiter plates. The plates were then incubated at 37 ºC for 24 h in 5 % CO 2 atmosphere. After incubation, 20 µl of MTS solution was added to each well and incubated for 3-4 h and OD was read at 490 nm. Cells without compounds served as blank and cadmium as the positive control. The experiment was carried out in triplicates.

Statistical analysis
Data reported were mean ± standard error of the mean. The statistical analysis was carried out using one-way analysis of variance (ANOVA) using Graphpad Instat version 3.0 software.

Antifungal activity
The aqueous extract and protein fraction (AMP III) isolated from the leaves of Atlantia monophylla Linn (Rutaceae) leaves exhibited antifungal activity on C. albicans and A. fumigatus. Using disc diffusion method, C. albicans exhibited 19 mm of inhibitory zone at 500 µg/ml concentration whereas A. fumigatus exhibited 11 mm at the same concentration. AMP III exhibited more antifungal activity than the crude aqueous extract. The aqueous extract showed minimum inhibition concentration on C. albicans at 125 and on A. fumigatus at > 200 µg/mL using micro broth dilution method. Out of four fractions (designated as AMP I, AMP II, AMP III and AMP IV) collected from ammonium sulphate protein precipitation method, only AMP III exhibited antifungal activity. AMP I, II, and IV did not exhibit activity on both strains of fungi ( Table 1). AMP III fraction exhibited 22 mm inhibitory zone on C. albicans and 16 mm on A. fumigatus.
SDS-PAGE was performed to identify the proteins in fraction III. Two protein bands were identified at 16 KDa and 67 KDa (Fig 1). The protein concentration was determined by using Bradford assay and found to be 240 and 670 µg/mL in crude extract and AMP III.

Antioxidant activity
The hydrogen atom or electron donation abilities of the corresponding extracts and some pure compounds were measured from the bleaching of the purple-colored methanol solution of 2, 2′diphenyl-1-picryl hydrazyl (DPPH). At 250 μg/mL concentration the aqueous extract of Atlantia monophylla Linn (Rutaceae) exhibited 79 % of DPPH radical scavenging activity and AMP III exhibited 90 % ( Table 2). Superoxide scavenging activity of both aqueous extract and AMP III were evaluated and the data obtained are presented in the Table 2. At 250 μg/ml concentration, the aqueous extract of A. monophylla Linn (Rutaceae) exhibited 69 % and fraction III exhibited 81 %. At 250 μg/ml concentration, the inhibition of H 2 O 2 scavenging activity of the aqueous extract of A. monophylla Linn (Rutaceae) was 82 % and AMP III exhibited 91 % as shown in Table 2.  The inhibition of ABTS scavenging activity was found to be 79 % in the aqueous extract of A. monophylla Linn (Rutaceae) and 94 % in AMP III at 250 µg/ml concentrations (Table 2). Cytotoxicity was found to be greater than 100 µg/ml (IC 50 ) for both the aqueous and AMP fraction III when tested on Vero cell lines. Compared to the ethanol extracts of A. monophylla Linn (Rutaceae) reported by Reddy et al [14], the aqueous and protein fraction exhibited greater activity in both antifungal and antioxidant activities.

DISCUSSION
From the results it is evident that the crude extract and the protein fraction (AMP III) extracted from the leaves of A. monophylla Linn (Rutaceae) possess good antifungal and antioxidant activities. With the rise in the incidence of opportunistic fungal infections, attempts are being made to develop new chemotherapeutic agents for treatment and management. Medicinal plants possess a mixture of compounds of known therapeutic properties [15]. Natural peptides/proteins or their analogs have been found with varying activities against pathogenic fungi with potential pharmaceutical utility [16]. Holetz et al [17] classified the potential of plant extracts and fractions based on MIC as potent (< 100 µg/ml), moderate (ranging from 100-500 µg/ml), weak (500 -1000 µg/ml) and inactive (> 1000 µg/ml). From the results it is evident that the crude aqueous extract is moderately active (ranges from 125 -250 µg/ml) and the AMP III fraction is very active (50 -90 µg/ml) on fungal strains as shown in Table 1. DPPH is a stable free radical at room temperature and accepts an electron or hydrogen radical to become a stable diamagnetic molecule. The reduction capability of DPPH radicals was determined by the decrease in its absorbance at 517 nm.
Sanchez-Moreno et al [18] classified the kinetic behavior of antioxidants as follows: < 5 min (rapid), 5 -30 min (intermediate) and > 30 min (slow). Based on this kinetics, both the crude aqueous and AMP III fraction exhibited radical scavenging activity differing in percentage activity. It is well known that superoxide anions damage biomolecules directly or indirectly by forming H 2 O 2 , .OH, peroxy nitrite or singlet oxygen during aging and pathological events such as ischemic reperfusion injury. Superoxide has also been observed to directly initiate lipid peroxidation [19].
Protein fraction III partially purified from A. monophylla Linn leaves exhibited greater scavenging activity than the crude extract. Hydrogen peroxide is a weak oxidizing agent and can inactivate a few enzymes directly, usually by oxidation of essential thiol (-SH) groups. Hydrogen peroxide can cross cell membranes rapidly, once inside the cell, H 2 O 2 can probably react with Fe 2+ , and possibly Cu 2+ ions to form hydroxyl radical and this may be the origin of many of its toxic effects [11]. It is therefore biologically advantageous for cells to control the amount of hydrogen peroxide that is allowed to accumulate.
ABTS is a relatively stable free radical which involves the direct generation of ABTS radical mono cation without any involvement of intermediary cation. Here, the radical cation is formed prior to addition of the antioxidant test system, rather than the generation of the radical taking place continually in the presence of antioxidant. This method, used for the screening of antioxidant activity, is applicable to both lipophilic and hydrophilic antioxidants [20]. The free radicals were scavenged in a concentrationdependent manner.

CONCLUSION
From the results it is evident that both the crude aqueous and protein fraction extracted from the leaves of Atlantia monophylla Linn possess both antifungal and antioxidant activities. Individual proteins from the protein fraction AMP III thus need to be isolated, purified, characterized and further evaluated for in vitro and in vivo antifungal and antioxidant activity.