Screening, Purification and Characterization of Anionic Antimicrobial Proteins from Foeniculum Vulgare

Foeniculum vulgare Mill., commonly called fennel, is a medicinal plant belonging to the Umbelliferae (Apiaceae) family, and is used in traditional medicine. Antibacterial peptides were isolated using sodium phosphate citrate buffer and, for extraction, cetyltrimethyl ammonium bromide (CTAB) buffer with pH 6, have been employed and antimicrobial activity tested against four reference strains. The extracted protein was subjected to 3 kDa dialysis and separation was carried out by DEAE-ion exchange chromatography and further proteins were identified by 2D gel electrophoresis. The results of Foeniculum vulgare elutes obtained from DEAE-ion exchange chromatography were tested for antibacterial activity. Elute 3 shows the highest antibacterial activity on Pseudomonas aeruginosa with a diameter of a zone of inhibition of 16 mm and IC50 value 25.02 (mcg/mL). Based on the findings of the wide usage in treatment of various ailments and day-to-day life, Foeniculum vulgare seeds were used in the present research and have shown promising antibacterial effects, which requires further proteomic research to authenticate the role of the anticipated proteins.


Introduction
Voluminous plants and plant extracts have a wide variety of resistance mechanisms to chemical, physical, and biological strains, like heavy metals, cold, drought, pathogens and contaminant attacks from bacteria, fungi, and viruses. Several plants exhibited complete genes, allied together for assimilated control over the infections from numerous types of pathogens [1]. Typically, the resistance control is accomplished by the discharge of secondary metabolites, such as phytoalexins, polyphenolics, and tannins, and the generation of pathogenesis-related proteins. In the early 1970s pathogenesis-associated proteins were primarily revealed from the leaves of tobacco. In response to the infections of tobacco mosaic virus were later well-defined as the induced proteins that are discharged through the pathogenic attacks [2].
Antimicrobial peptides are pervasive and the established host resistances, in contrast to pathogens and pests in different organisms, vary from microorganisms to animals [3]. Antimicrobial peptides occur in various molecular arrangements, while most of them are linear peptides from plants, insects,

Results and Discussion
The protein extract after extracting in sodium acetate buffer, pH-6.5, were purified by dialysis, and the same extract was concentrated and subjected for antibacterial testing against four standard pathogenic bacterial strains found to cause foodborne illness and spoilage of food and herbal drugs. The bacterial strains used in the study were Escherichia Coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), and Proteus vulgaris (P. vulgaris), and compared with the standard antibiotics ciprofloxacin (25 mcg/mL) and chloramphenicol (100 mcg/mL).

Comparison of Protein Concentration Extracted in Different Buffers after Dialysis
The result of the study shown in Table 1 reveals that different buffers and pH vary in the concentration of protein extracts. Seeds of Foeniculum vulgare Mill., showed low concentration of protein, at 80 µg/mL, when extracted in sodium phosphate citrate buffer (pH-7.2) and CTAB buffer (pH 6.0), and exhibited the highest concentration of protein, at 140 µg/mL, with sodium acetate buffer (pH-6.5). Antibacterial activity results shown in Table 2 and Figure 1 were obtained from sodium phosphate citrate buffer and CTAB buffer extracts from Foeniculum vulgare Mill. (S1) were found to exhibit nil activity on all of the bacterial strains used, so this plant's seeds were again extracted in sodium acetate buffer, pH-6.5, to obtain a sensitivity pattern on the bacterial strains. Pseudomonas aeruginosa exhibited very good sensitivity when compared with the other bacterial strains with a diameter of the zone of inhibition 12.5 mm, whereas Staphylococcus aureus and Proteus vulgaris showed similar zones of inhibition of 12 mm. E. coli, Pseudomonas aeruginosa, and Proteus vulgaris revealed good zone of inhibition patterns of 11 mm, 12.5 mm, and 12 mm, when compared with the standard antibiotic Chloramphenicol (25 mcg/mL), which is only 8 mm.

Antibacterial Activity of Sodium Acetate Buffer pH-6.0 Extracts after Dialysis
Antibacterial activity results shown in Table 2 and Figure 1 were obtained from sodium phosphate citrate buffer and CTAB buffer extracts from Foeniculum vulgare Mill. (S1) were found to exhibit nil activity on all of the bacterial strains used, so this plant's seeds were again extracted in sodium acetate buffer, pH-6.5, to obtain a sensitivity pattern on the bacterial strains. Pseudomonas aeruginosa exhibited very good sensitivity when compared with the other bacterial strains with a diameter of the zone of inhibition 12.5 mm, whereas Staphylococcus aureus and Proteus vulgaris showed similar zones of inhibition of 12 mm. E. coli, Pseudomonas aeruginosa, and Proteus vulgaris revealed good zone of inhibition patterns of 11 mm, 12.5 mm, and 12 mm, when compared with the standard antibiotic Chloramphenicol (25 mcg/mL), which is only 8 mm.

Protein Concentration of Ion Exchange Chromatography Elutes
The crude protein extracts from Foeniculum vulgare Mill. extracted in sodium acetate buffer at a pH of 6.5 after dialysis were further purified using the DEAE-ion exchange chromatography technique to isolate and characterize the antibacterial proteins present in the extract. The crude protein extract after dialysis was subjected to ion exchange chromatography, four elutes were obtained, and the concentration was found using the Lowry method. The protein concentration was found in ranges from 100-120 mcg/mL, the highest concentration was seen in elute 1 and 2 with 120 mcg/mL, and the least was found in elute 4, with 100 mcg/mL, results shown in the Table 3.

Protein Concentration of Ion Exchange Chromatography Elutes
The crude protein extracts from Foeniculum vulgare Mill. extracted in sodium acetate buffer at a pH of 6.5 after dialysis were further purified using the DEAE-ion exchange chromatography technique to isolate and characterize the antibacterial proteins present in the extract. The crude protein extract after dialysis was subjected to ion exchange chromatography, four elutes were obtained, and the concentration was found using the Lowry method. The protein concentration was found in ranges from 100-120 mcg/mL, the highest concentration was seen in elute 1 and 2 with 120 mcg/mL, and the least was found in elute 4, with 100 mcg/mL, results shown in the Table 3. Table 3. Comparison of the protein concentration (mcg/mL) after ion-exchange chromatography.

Antibacterial Activity
The results of Foeniculum vulgare elutes obtained after ion exchange chromatography were tested for antibacterial activity as shown in Table 4 and Figure 2. Elute 3 shows the highest antibacterial activity on P. aeruginosa with a diameter of the zone of inhibition of 16 mm and an IC 50 value of 25.02 mcg/mL, when compared with the standard antibiotic Chloramphenicol at 100 mcg/mL which is only 12 mm and has an IC 50 value of 14.634 mcg/mL. Elute 2 shows a good zone of inhibition on Proteus vulgaris with a diameter of the zone of inhibition of 13 mm and an IC 50 value of 57.83 mcg/mL, and the lowest zone of inhibition was seen on P. aeruginosa with a zone of inhibition of 4 mm and an IC 50 value of 68.33 mcg/mL. S. aureus was found to shown good sensitivity toward elute 4 with a diameter of the zone of inhibition of 12 mm and an IC 50 value of 20.8 mcg/m) and, with elute 1, a diameter of the zone of inhibition of 10 mm and an IC 50 value 27.64 mcg/mL. E. coli exhibited good sensitivity towards elute 1 with a zone of inhibition at 13 mm and IC 50 value of 67.56 mcg/mL.

Antibacterial Activity
The results of Foeniculum vulgare elutes obtained after ion exchange chromatography were tested for antibacterial activity as shown in Table 4 and Figure 2. Elute 3 shows the highest antibacterial activity on P. aeruginosa with a diameter of the zone of inhibition of 16 mm and an IC50 value of 25.02 mcg/mL, when compared with the standard antibiotic Chloramphenicol at 100 mcg/mL which is only 12 mm and has an IC50 value of 14.634 mcg/mL. Elute 2 shows a good zone of inhibition on Proteus vulgaris with a diameter of the zone of inhibition of 13 mm and an IC50 value of 57.83 mcg/mL, and the lowest zone of inhibition was seen on P. aeruginosa with a zone of inhibition of 4 mm and an IC50 value of 68.33 mcg/mL. S. aureus was found to shown good sensitivity toward elute 4 with a diameter of the zone of inhibition of 12 mm and an IC50 value of 20.8 mcg/m) and, with elute 1, a diameter of the zone of inhibition of 10 mm and an IC50 value 27.64 mcg/mL. E. coli exhibited good sensitivity towards elute 1 with a zone of inhibition at 13 mm and IC50 value of 67.56 mcg/mL.   The analysis was performed using SAS for Windows version 9.2 (SAS Institute Inc., Cary, NC, USA). One-way ANOVA was used to compare the IC 50 of different elutes on four bacterial strains used in the study. Among the tested strains, S. aureus and P. vulgaris showed the highest significant inhibition in comparison to standard cipro (p = 0.02) whereas there is no significance observed in the IC 50 in E. coli and P. aeruginosa compared to standard cipro, results shown in Table 5.

2-D Gel Electrophoresis Results
The purified proteins from Foeniculum vulgare seeds after anion exchange chromatography were subjected to 2-DE. Out of four elutes obtained from anion exchange chromatography, only elute 2 was used for 2-DE analysis. The elute 2 protein sample extracted from Foeniculum vulgare seeds was analyzed to investigate the proteins present in the sample based on the molecular weight, using HP Scanjet G4010 proteomic techniques. The analysis of 2D gel of Foeniculum vulgare showed reproducible protein spots, most of which were distributed near the center of the gels, between pI values 5-8 and molecular weight of 34.4 kDa and 48 kDa (Figure 3).  The analysis was performed using SAS for Windows version 9.2 (SAS Institute Inc., Cary, NC, USA). One-way ANOVA was used to compare the IC50 of different elutes on four bacterial strains used in the study. Among the tested strains, S. aureus and P. vulgaris showed the highest significant inhibition in comparison to standard cipro (p = 0.02) whereas there is no significance observed in the IC50 in E. coli and P. aeruginosa compared to standard cipro, results shown in Table 5.

2-D Gel Electrophoresis Results
The purified proteins from Foeniculum vulgare seeds after anion exchange chromatography were subjected to 2-DE. Out of four elutes obtained from anion exchange chromatography, only elute 2 was used for 2-DE analysis. The elute 2 protein sample extracted from Foeniculum vulgare seeds was analyzed to investigate the proteins present in the sample based on the molecular weight, using HP Scanjet G4010 proteomic techniques. The analysis of 2D gel of Foeniculum vulgare showed reproducible protein spots, most of which were distributed near the center of the gels, between pI values 5-8 and molecular weight of 34.4 kDa and 48 kDa. Antibiotic resistance is one the most dangerous global problems today, which makes antimicrobial proteins/peptides the agents of a substitute for cures of pathogenic diseases [23]. In this study, we have extracted 3 kDa antimicrobial peptides from Foeniculum vulgare Mill. seeds using buffers and the specific proteins of interest were purified with a dialysis technique using 3 kDa dialysis tubing. Conversely, further separation was done using DEAE-ion exchange Antibiotic resistance is one the most dangerous global problems today, which makes antimicrobial proteins/peptides the agents of a substitute for cures of pathogenic diseases [23]. In this study, we have extracted 3 kDa antimicrobial peptides from Foeniculum vulgare Mill. seeds using buffers and the specific proteins of interest were purified with a dialysis technique using 3 kDa dialysis tubing. Conversely, further separation was done using DEAE-ion exchange chromatography and four elutes were subjected to antimicrobial activity, out of which elute 2 has promising results, which was then characterized by using 2D gel electrophoresis. Much expected optimistic results were obtained against tested pathogenic bacteria, where they exhibited antibacterial activity in comparison to the standard cipro (100 mcg). Past studies have demonstrated that asymptomatic bacteriuria posed more serious hazards in pregnant women [24], so bacterial resistance is inciting a resurgence in research of the antimicrobial activities of herbs against resistant strains [25,26].
In the present research work on anion exchange resin diethyl amino ethyl cellulose (DEAE) was used, which is positively-charged to exchange negatively-charged protein. DEAE-ion exchange chromatography is one of the promising techniques used to separate proteins based on their surface ionic charge using resins that are altered with either negatively-charged or positively-charged biochemical groups [27]. As in this study, anion exchange resin DEAE is being used, as proteins purified by ion exchange chromatography may contain a negative charge.
Proteomics is a handy technique used in the characterization of various proteins [28]. Additionally, 2-DE polyacrylamide gel electrophoresis is a potent technique and, as such, can be adopted to separate and determine protein mixtures of thousands of specific components [29], is based on the gel size, is capable of determining more than 5000 proteins at once, and can detect with less than 1 mg of sample per spot. In our research work, 2-D PAGE is used for the characterization of anionic proteins from elute 2 of the ion-exchange chromatography sample of Foeniculum vulgare seed extract, and most of the protein spots were found to be between pI values 5-8 and molecular weights of 34.4 kDa and 48 kDa on the 2D gel, which was found to be higher in molecular weight and possess antibacterial activity from being tested on the four bacterial strains. Previous study identified napins, a class of proteins from 2S albumin, which were isolated from plants and that can be easily extracted from seeds [30] which are composed of 10 kDa and 4.5 kDa subunits [31]. Furthermore the present study has provided more evidence that the high mass protein isolated from the Gram-negative plant pathogen, and also more researchers' stated literature reports on carotovoricin [32] and colicins [33][34][35], authenticating our present research study.

Materials
The Foeniculum vulgare Mill. seeds were procured from a local market in Riyadh, Saudi Arabia. Before adopting the protocol, the plant seeds were identified by Dr. Ali S. Alqahtani, Pharmacognosy Department, King Saud University, Riyadh, Saudi Arabia. Ethical approval was obtained from the research ethics committee (CAMS-153-36/37) from the college of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia.

Preparation of Plant Seeds Extract
The Foeniculum vulgare Mill. seeds were cleaned using sterile water to remove any debris left over on the seeds and dried in a biological safety cabin to maintain the sterile condition. Later, fine powder was prepared from the seeds under aseptic conditions and the extract was soaked in sodium phosphate buffer (pH-6.5) at 30 • C and left for overnight incubation. The next day, extracts were filtered with Whattmann filter paper No. 1 and subjected to 80% ammonium sulfate saturation. The collected saturated material was then separated by dialysis using a 3 kDa cutoff dialysis tubing (Sigma Aldrich, St. Louis, MO, USA) and the protein' concentration was further estimated by spectroscopic analysis at a wavelength of 280 nm.

DEAE-Ion Exchange Chromatography
The chromatography column was prepared using a DEAE cellulose bed of 1 cm thickness, washed with ethanol before use, 20 mM Tris-Hcl for the 250 mL required volume was prepared, and pH was attuned to 8.5 using NaOH. Bed preparation: counter ions (salt gradient) of 40 mL of 25 mM NaCl in 50 mM tris (pH-7.2) and 0.3% of DEAE were applied to the column. The elutes were run for 4 h and 2 mL was collected. Each column used a flash chromatography column from Sigma Aldrich, St. Louis, (MO, USA), and the eluting buffer was sodium phosphate citrate buffer (pH-7.2). Elute preparation: four elutes were prepared in test tubes for each dialyzed sample. After dialysis, the samples were poured into the column without disturbing the bed, and left for 20 min to settle. The first eluting buffer, i.e., sodium phosphate citrate buffer (pH-7.2), was loaded into the column and was used to elute the sample. Thed column was allowed to settle for 15-20 min. The flow rate is adjusted to 1 mL/min and the elute was collected in test tubes at the bottom.

Bacterial Strains
Bacterial strains used for the study were E. coli, P. aeruginosa S. aureus, and P. vulgaris, and were obtained from department of laboratory sciences, College of Applied Medical Sciences, Riyadh, Saudi Arabia.

Culture Medium and Inoculum Preparation
Antibacterial activity of this crude extract was tested against these four strains in Hi-media. Culture slants were prepared by taking a loop full of pure culture and dispensing into 10 mL nutrient broth from four strains in a sterilized laminar flow and incubated for two days in the incubator. The final concentration of each active culture was maintain at 108 cfu/mL.

Agar Well Diffusion Assay
The antibacterial activity of the crude protein extracts was determined by agar well diffusion assay [36]. Pure cultures were dispersed on agar plates using sterile cotton swabs. Next, using a sterile borer, wells were created on the agar plates. Crude protein extracts (100 µL) filled the wells, with antibiotic and buffer as negative controls, and incubated for a day in an incubator. All of the plates were conducted in triplicate to ensure accurate results [37] and the zone of inhibition was measured in millimeters.

Determination of the Minimal Inhibitory Concentration (MIC)
The protein extracts of different plant seeds were tested for antibacterial activity against four strains by the micro-dilution method [38], with some alterations and suggestions by NCCLS (NCCLS, 2001). Different concentrations of extracts were loaded in 96-well plates to check the antibacterial activity of the extracted proteins. The suspension of culture with 10 8 cfu/mL concentration added to each well and made a final volume to 200 µL by adding LB broth. Ten microliters of MTT (5 mg/mL) was added to all of the wells after incubation at ±4 • C for 18 h and the readings were taken at 530 nm using an ELISA reader (Tecan, Männedorf, Switzerland). The minimum concentration of protein that showed total inhibition was considered as its MIC minimum zone of inhibition. As control experiments, Chloramphenicol (25 mcg) and Ciprofloxacin (100 mcg) were taken as positive controls, and sterile broth as the negative control.

Statistical Analysis
The inhibition concentration at 50% inhibition (IC 50 ) was the parameter used to compare the antibacterial activity. A lower IC 50 means better antibacterial activity. All of the tests were performed in triplicate and the results were expressed as the mean ± standard deviation. The percent of inhibition of antibacterial activity of the protein was calculated based on: % Inhibition = (A control − A sample)/A control × 100.

Two-Dimensional Electrophoresis
Two-dimensional (2-D) electrophoresis, one the most important tool in proteomics, was performed in accordance with the method reported in [29]. The sample was dissolved in 120 µL of rehydration buffer, loaded onto the 17 cm IEF strip 3-10 pH Liner, (Biorad, Hercules, California, CA, USA), and retained for iso-electric focusing. Next, the strips were equilibrated in equilibrium buffer after finishing the IEF run and the second dimension was run on 12% SDS PAGE.

Conclusions
Over a long period of time, Foeniculum vulgare seeds have shown promise in traditional medicine for the treatment of a wide range of diseases. From our results, 14 spots from the 2D gel were eluted and further subjected to mass spectrometry studies, undergoing more proteomic analyses and the isolation of small peptides from Foeniculum vulgare. Many studies have reported that this plant has numerous in vivo and in vitro pharmacological assets that can be used in various aspects of prognosis. Based on the findings of the wide usage in treatment of various ailments and day-to-day life, Foeniculum vulgare seeds were used in the present research and have established the promising antibacterial effects of these seeds, which require further proteomic research to authenticate the role of anticipated proteins.