ISOLATION OF ANTIBACTERIAL PEPTIDE FROM VIGNA ACONITIFOLIA L.

ISOLATION OF ANTIBACTERIAL PEPTIDE FROM VIGNA ACONITIFOLIA L.

D. Swathi * and N. Mallikarjun

Department of Studies and Research in Microbiology, Sahyadri Science College, Kuvempu University, Shivamogga, Karnataka, India.

ABSTRACT: Vigna aconitifolia L. represents a genus in the Fabaceae family and is one of the most drought-resistant legumes, commonly grown in arid and semi-arid regions of India. Therapeutic effects of Vigna aconitifolia L. have been reported in several publications, but there are only a few reports on the biological activities of the Vigna aconitifolia L. proteins. In the present study, antibacterial activities of the Vigna aconitifolia L., peptides have been investigated. Homogenized seed flour was pulverized, and its protein content was extracted in a phosphate buffer. After centrifugation, by adjusting pH, peptides from the supernatant were isolated. Gel filtration (G-25) and Ion-exchange chromatography on the seed extract gave fairly pure peptide fractions of which inhibitory activities were done by agar well diffusion method against Shigella flexineri, Bacillus cereus, Salmonella typhimurium, Staphylococcus aureus, Enterococcus faecalis, Vibrio cholerae bacterial pathogens. The results indicate that a 30kDa of the peptide was identified and exhibited good antibacterial activity against bacterial pathogens. Among all strains, Shigella flexineri and Staphylococcus aureus exhibit good results with a clear zone of inhibition.

Keywords: Antibacterial, Chromatography, Drought-resistance, Peptides, Pulverized

INTRODUCTION: Antibacterial peptides (ABPs) are part of antimicrobial peptides with small glycine, and cysteine-rich amino acid residues of polypeptides known as “host defense peptides” and a part of the innate immune response found among all classes of life ^1-9. Antibacterial peptides have a molecular weight of 4-50kDa, which plays a role in defense of the plant immune system and inhibits various bacterial species ^{2, 5}. In the past two decades, a considerable increase in resistance in pathogenic bacteria against common antibiotics has emerged.

Those bacteria, such as Escherichia coli, Salmonella sp, and Klebsiella sp, although they were susceptible to the usual antibiotic therapy some time ago, have now made the main cause of challenging efforts in treating nosocomial infections ³. Rising bacterial resistance faster against synthetic antibiotics has turned attempts to search for antibiotics from natural sources, again ¹¹. Among different categories of natural substances, proteinaceous materials isolated from plant sources have given promise in finding new natural compounds with good antimicrobial activities.

In this way, many small proteins with some activity against microbial pathogens have been identified in plant seeds ¹⁴. Vigna aconitifolia L. represents a genus in the Fabaceae family (sub-family Papilionaceae). Vigna aconitifolia L. in generalis known as mat bean, moth bean, matki, Turkish gram, or dew bean. It is one of the most drought-resistant legumes, commonly grown in arid and semi-arid regions of India. Itsfamine-resistant merits made it fight soil erosion. With its high protein content, the Vigna aconitifolia L. has been identified as possibly a more significant food source in the future. Therapeutic effects of Vigna aconitifolia L. have been reported in several publications, but there are only a few reports on the biological activities of the Vigna aconitifolia L. proteins ⁴. The present study used chromatographic approaches to extract and purify antibacterial peptides from Vigna aconitifolia L. seeds.

MATERIALS AND METHODS:

Sample Collection: The two varieties [V1 and V2] Vigna aconitifolia L. seeds were obtained from the local market in Dharwad, Karnataka. The seeds collected were identified and authenticated in the Department of Studies and Research in Botany, Sahyadri Science College, Shivamogga, Karnataka. India.

Bacterial Strains: All the bacterial strains used in this study were collected from the National Collection of Industrial Microorganisms (NCIM), Pune, India, and Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology (IMTECH), Chandigarh, India. The bacterial strains include Staphylococcus aureus (NCIM 2073), Enterobacter faecalis (NCIM 5253), Salmonella typhimurium (NCIM 2501), Bacillus cereus (NCIM 2217), Vibrio cholerae (MTCC 2906), Shigella flexineri (NCIM 5265). The cultures were preserved at 4^oC and subcultured at regular intervals.

Extraction and Purification of the Moth Bean Seeds: Fresh seeds of moth bean were collected and rinsed with 0.1% of sodium hypochlorite solution for 10 min and then washed with distilled water for the removal of dirt and debris. Seeds were dried under sterile conditions and pulverized into fine powder; 30 gm of seed sample was homogenized with 100 ml of 0.1 M Phosphate buffer (pH 7). The homogenate was then centrifuged at 6000 rpm for 15 minutes at 4 ℃. The supernatant thus obtained was used as the crude peptide extract. The obtained crude extract was saturated to 40% and 80% relative ammonium sulfate precipitation by adding solid ammonium sulfate (12.6 g) with constant stirring at 4 ℃. This solution was incubated overnight at 4 ℃. The solution was centrifuged at 8500 rpm for 15 minutes at 4℃. The obtained pellets were re-dissolved in 10 ml of 10mM Tris buffer and stored at 4 ℃. The resulting suspension was extensively dialyzed against distilled water. The dialyzed solutions were recovered and subjectedto further purification by chromatographic methods ¹³.

Gel Filtration Chromatography: The column filled with Sephadex G-25 pre-equilibrated with 0.1 M phosphate buffer (pH 7) was initially used to separate proteins from the F/3-10 fraction. The salt-precipitated sample (80 %) was loaded in the column and 40 ml of 0.1 M phosphate buffer (pH 7) was added to it. The fractions of 1 ml volume were collected at the flow rate of 1 ml/5mins. All the collected fractions were assessed for protein content by 280nm methods ¹⁴.

Ion Exchange Chromatography: The fractions showing the highest protein content (fractions 3-10) were pooled and used for Ion exchange chromatography ⁶, DEAE- Sephadex column pre-equilibrated with 0.025M Tris- NaCl buffer. The fractions were prepared at different ionic strengths, these fractions were collected at the flow rate of 1 ml/5min, and a stepwise increase in ionic strength eluted the protein. The amount of protein in these fractions was estimated by Lowry’s method using Bovine serum albumin (BSA) as the standard ⁷. 

SDS-PAGE: The purification process was monitored throughout by SDS-PAGE, performed according to the method described by Laemmle ⁸. The sample buffered contained (Tris-HCl 6g, Glycine 14.4g, SDS 1g, distilled water- 1000ml, pH-8.2). After the run, the gel was removed from plates and put in staining solution (40ml methanol, 5ml acetic acid, 55ml distilled water, 0.01g Coomassie brilliant blue dye) overnight and later destained with destaining solution (40ml methanol, 5mL acetic acid, 55mL distilled water) until the bands were visible.

Antibacterial Assay: Antibacterial activity of the crude and purified peptide concentrates from seed sample was determined using Agar well diffusion method ¹³ against bacterial pathogens such as Staphylococcus aureus, Enterobacter faecalis, Salmonella typhimurium, Bacillus cereus, Vibrio cholerae, Shigella flexineri. The test organism (100ul) was inoculated on the surface of the Nutrient Agar medium. Isolated peptides of different concentrations (100ul, 150ul, 200ul) were loaded onto each well and incubated at 37^oC for 24 h. The zone of inhibition was measured, and the assays were performed in triplicates.

RESULTS: The present study was carried out to extract, purify, and isolate antibacterial peptides from Vigna aconitifolia L. seeds.

Spectrometric Determination of Protein: The concentration of total protein content present in the seeds is shown in Table 1. The protein concentration of moth bean in crude extract and purified elutes were represented in mg/100gm of seeds. Both varieties of moth bean show the highest protein content thus, it is further selected for antibacterial activity.

Antibacterial Activity of Crude Peptide Extract: The antibacterial activity of the crude peptide concentrates from seeds of moth beans was determined by Agar well diffusion assay. Both varieties (V-1 and V-2) exhibit good inhibitory activity against six pathogenic bacteria Table 2.

Data represents that among all strains, Shigella flexineri and Staphylococcus aureus exhibit good results with a zone of inhibition of 20±1.24 and 21.6±1.44 Fig. 1, respectively followed by Enterobacter faecalis 15±1.05, Salmonella typhimurium 12±1.05, Bacillus cereus 10±1 Vibrio cholera 10±0.5 even at low concentration (100μl/well).

FIG. 1: ANTIBACTERIAL ACTIVITY OF CRUDE PEPTIDE AGAINST BACTERIAL STRAINS [A] Shigella flexineri and [B] Staphylococcus aureus. In the figure: A=100 μl,B=150 μl,C=200 μl,D=Standard antibiotic.

TABLE 1: PURIFICATION STAGES OF PEPTIDES FROM VIGNA ACONITIFOLIA L (V-1 AND V-2)

 TABLE 2: ANTIBACTERIAL ACTIVITY OF CRUDE PEPTIDE ISOLATED FROM THE SEED OF VIGNA ACONITIFOLIA L.(V-1 AND V-2) AT DIFFERENT CONCENTRATIONS

Where “-” shows, inhibition not detected, all values were expressed in ± Standard Error Mean

 Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis: Peptide fractions obtained were subjected to SDS-PAGE. The migration of molecular mass standards was indicated in kDa on the left-hand side. Based on Lowry’s method ⁷ of protein estimation results, the total amount of peptide was determined, and 33μg of peptide sample was loaded on wells. The molecular profiling of the sample was observed and indicated the presence of a peptide with a molecular weight ranging from 16 kDa to 175kDa. According to the previously reported studies, the peptide bands with molecular weight 30kDa Fig. 2 distinguished in this study were used for further activity.

TABLE 3: ANTIBACTERIAL ACTIVITY OF ELUTED PEPTIDE ISOLATED FROM THE SEED OF VIGNA ACONITIFOLIA L(V-1 AND V-2) AT DIFFERENT CONCENTRATIONS

Where “-” shows, inhibition not detected, all values were expressed in ± Standard Error Mean

Antibacterial Activity of Eluted Peptide Sample: To check the antibacterial activity of eluted peptide band, the peptide was eluted from an elution buffer, further inhibitory activity was done against six bacterial pathogens. As shown in Fig. 2. A shows a clear zone of inhibition of 19±1.24 at 150 μl of eluted peptide content and 20±1.24 at 200 μl of eluted peptide content against bacterial pathogen Shigella flexineri. [B] shows a zone of inhibition of 14±0.5 mm with 150 μl of eluted peptide content and 18±1.05 at 200 μl of the peptide against Staphylococcus aureus, the negative control is Tris-HCl (pH=8.8).

FIG. 2: ANTIBACTERIAL ACTIVITY OF ELUTED PEPTIDE AGAINST BACTERIAL STRAINS [A]Shigella flexineri and [B]Staphylococcus aureus. In the figure: A=100 μl,B=150 μl,C=200 μl,D=Standard antibiotic.

FIG. 3: SDS-PAGE ANALYSIS OF PEPTIDE EXTRACT: 1. SHOWS THE MOLECULAR MARKER, 2. CRUDE EXTRACT 3. PURIFIED PEPTIDE

Statistical Analysis: All the experiments were carried out in triplicates (n=3). The statistical analysis of the data was carried out by analysis of the variance (ANOVA). Results were considered significant when p<0.05.

DISCUSSION: This study described the isolation of antibacterial peptides from Vigna aconitifolia L; we found that the isolated peptide exhibited strong antibacterial activity against gram-positive and gram-negative bacteria that as Staphylococcus aureus and Shigella flexineri with a goodinhibition zone. In the previous study, antimicrobial peptides with gram-positive and gram-negative bacteria growth-inhibiting abilities have been reported ⁹. Numerous molecules with antibacterial activity have been isolated from plants ^{10, 12}. According to the World Health Organization (WHO), medicinal plants would be a very effective source of obtaining a variety of active compounds and drugs. Therefore, such plants should be investigated to understand their properties, efficiency, and safety ¹⁶. Hence an attempt is made to explore the possible antimicrobial peptides generated through the medicinal plants ¹⁷. In previous studies, the antibacterial peptide was isolated from different plant sources. Similar to our study, we found potential antibacterial peptides isolated from capsicum ¹⁵. Several antimicrobial peptides have been reported ¹⁷, purified and characterized by using different techniques like ion exchange chromatography and RP-HPLC, which are quite expensive and time-consuming ¹⁸. Here, we have isolated the antibacterial proteins/peptides from Vigna aconitifolia L by cost-effective and time-saving methods for plant protection and therapeutic potential.

CONCLUSION: It is concluded that traditional plants may represent new sources of antimicrobials with the use of AMPs as single therapeutic antibiotic agents. The current study identified a novel antibacterial peptide from Vigna aconitifolia L. The results showed that 30kDa Fig. 3 of the peptide was identified and exhibited broad-spectrum antibacterial activity, which can be further used to develop novel therapeutic agents and biologically active components. Further Proteomic analysis is in progress to ensure the authenticity of the results regarding the peptide activity tested in our study.

ACKNOWLEDGEMENT: We are grateful to the Department of Microbiology, Sahyadri Science College, Shimoga, Karnataka, for supporting us in this work. We take the privilege to acknowledge Dr. N. Mallikarjun, Department of Microbiology, Sahyadri Science College, Shimoga, Karnataka, for his contribution to the project and manuscript preparation. We are thankful to the Department of OBC for a financial assistantship.

CONFLICTS OF INTEREST: The authors declare that they do not have any conflict of interest.

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    How to cite this article:

    Swathi D and Mallikarjun N: Isolation of antibacterial peptide from Vigna aconitifolia L.. Int J Pharm Sci & Res 2023; 14(5): 2477-82. doi: 10.13040/IJPSR.0975-8232.14(5). 2477-82.

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