Structural Features and Mechanism of Action of AMPs Generally, AMPs are short peptide molecules having 10-50 amino acid residues. Most of them contain basic amino acids in their structure, therefore they are cationic in nature showing a net positive charge ranging from +3 to +11. Due to positive charges, most of the AMPs can bind electrostatically with the negatively charged surface of the pathogen e.g. teichoic acid of gram-positive bacteria, lipopolysaccharide of gram-negative bacteria and cytoplasmic membrane of both gram-positive and gram-negative bacteria having negatively charged phospholipids. However, AMPs exhibit weak interactions with the mammalian cytoplasmic membrane as it contain mostly neutral phospholipids. As a result, they selectively and preferentially act against bacteria and other pathogens, while avoid binding with the mammalian cells. Due to this striking feature, AMPs have been considered as natural antibiotics as well as antimicrobial agents. Another important feature of AMPs is the presence of hydrophobic amino acid residues which control the hydrophobic binding and insertion behavior in the lipid membrane. In some AMPs, the cationic and hydrophobic residues are located in opposite direction, therefore, these peptides exhibit amphipathicity (Tossi et al., 2000). This amphipathic arrangement enables them to bind with both the hydrophilic and the lipophilic surface of the target membranes. Depending on secondary structure, AMPs can be divided into α-helix, β-sheet, mixed or random-coil structure. Several AMPs remain unstructured in aqueous solution but obtain a secondary structure or conformation (e.g., α-helix) in presence of lipid membranes. This conformational change sometimes plays significant role on the antimicrobial activity of AMPs.
The mechanism of action of AMPs differs greatly based on their target pathogens. For example, antiviral AMPs interfere with viral envelopes (Robinson et al., 1998; Sitaram and Nagaraj 1999), antibacterial and antifungal AMPs causes membrane permeabilization and inhibit the protein synthesis (Brogden 2005). The mechanism of antibacterial activity of AMPs has been extensively studied and it can be classified into two groups- one is membrane permeabilization which leads to cell lysis and cell death (Chan et al., 2006), another is blocking the synthesis of intracellular organelles e.g. enzymes or proteins.
Sources of Plant AMPs in Bangladesh Plants are good sources of antimicrobial peptides (AMPs), which provide defensive action against a wide range of microbes (Nawrot et al., 2014). Plant antimicrobial peptides can be classified as thionin, purothionin, defensin, lipid transfer protein, 2s albumin, napin, snakins, knottin-type, peptide so-d1, cycloteinases and glycine rich protein. These AMPs also could be isolated from Bangladeshi plants.
Variation of Plants AMPs Based on Sources AMPs are found in the different species of plant, animal, insects and microorganisms. A single AMP can be found in various sources and a single source may contain multiple AMPs. For example, defensin is present in Cucumis sativus, Nigella sativa, Dahlia pinnata whereas wheat (Triticum aestivum) contains several AMPs including purothionin, hevein-like peptides and defensin (Hussain et al., 2013; Odintsova et al., 2013). However, geographical variation may cause discrepancies in the structure, mode of action and activity of AMPs of the same species. Staphylococcus hominis (MBL AB63), a jute endophyte isolated from Bangladeshi jute seeds, exhibited significant antibacterial activity against Staphylococcus aureus SG511 (Uddin et al., 2021). Therefore, it is important to extract and evaluate the activity of different AMPs present in the Bangladeshi plants.
Extraction of AMPs Extraction process involves the isolation of soluble material from an insoluble residue using a suitable solvent system (organic and aqueous) on the basis of the physical nature of the compound to be extracted. Generally solid phase extraction (SPE) and chemical extraction processes have been used to isolate different class of plant AMPs. Before going to extraction phase, the different plant parts are homogenized using grinding or milling.
Solid phase extraction (SPE) Solid phase extraction (SPE) is a very popular extraction method that involves partitioning between a liquid sample matrix and a solid phase that can be used for extraction, amount enrichment, derivatization and compound fractionation. The SPE process can analyze samples which are in solution, free of contaminating matrix compounds and concentrated enough for detection. Solid phase extraction is used to concentrate and purify the analytes from solution by sorption on solid sorbent molecules and purification of extract after collection. The process can be used for the extraction of molecules from different matrices which includes physiological fluid, water, beverages, soil, and living tissues (wir-Ferenc and Biziuk 2006). For substances that are in a solution, ion exchange SPE can be utilized. The electrostatic interaction of the charged functional group of the compound to the charged group bound to the silica surface is the compound's major retention strategy (Font et al., 1993). However, pH in SPE is very crucial. Silica-based surfaces have a pH range of 2 to 7.5, and at pH levels above and below this range they are stable.
Chemical Extraction Generally, two types of solvents are used to extract AMPs from plant sources. One is water and buffer solutions containing Na2HPO4 (Disodium phosphate), NaH2PO4 (Monosodium phosphate), KCl (Potassium chloride), EDTA (Ethylene diamine tetra-acetic acid). The other solvent system includes organic-based solutions such methanol, ethanol, acetonitrile, methylene chloride, chloroform etc. Some plants contain very low amount of AMPs, therefore it is necessary to concentrate the AMPs solution. For this purpose, organic solvent extraction and ammonium sulfate precipitation method have been used. In case of ammonium sulfate precipitation method, the AMPs are concentrated by salting out approach. Then, concentrated AMPs are desalted by dialysis. After formation of the pellets of extractable materials, they are resuspended and finally dialyzed using a definite molecular weight cut-off dialysis tubing (Osborn et al., 1995; Barber 1988). Moreover, several organic solvents are used to extract and concentrate specific group of AMPs including cyclotides. However, these organic solvents also extract low molecular weight compounds other than peptides.