Table 1 Approaches to the isolation of different structural families AMPs from various plant material

Water and water-based solutions

 Water

New Cysteine-Rich Peptides

Potentilla anserina (roots)

1. Dried roots were extracted at room temperature for 1 h and centrifuged at 10,000 rpm for 20 min;

2. Concentration by adding ammonium sulfate to 80% relative saturation;

3. Desalting was performed using C₁₈ reversed-phase flash column at a stepwise gradient of ethanol (40, 60, 80%)

[24]

8-Cys hevein-like peptides

Moringa oleifera, (fresh leaves)

1. Plant material was extracted with an equal volume of water while blending (6 min). The mixture was centrifuged at 8000 rpm for 10 min;

2. The supernatant was filtered and loaded on C₁₈ flash-column, elution was performed using increase of ethanol concentration (20, 70%)

[41]

 Acid solution 50 mM H₂SO₄

Defensin

Nicotiana alata (flowers)

1. Flowers were ground with liquid nitrogen by mortar and pestle, then extracted with sulfuric acid (3 mL/g wet weight) for 1 h. Insoluble material was filtered and centrifuged (25,000g, 15 min, 4 °C)

2. pH of supernatant was adjusted to 7.8 by adding NaOH, and stirred for 1 h, then centrifuged (25,000g, 15 min, 4 °C)

3. Concentration by adding solid ammonium sulfate to 80% relative saturation (stirring for 4–16 h at 4 °C)

4. The precipitate was dissolved in gel-filtration buffer, heated to 90 °C and separated using Sephadex G-50 gel-filtration column

[42]

 Acid mixture: 1% v/v trifluoracetic acid (TFA), 1 M HCl, 5% v/v formic acid, 1% w/v NaCl

Gly/His-rich peptides

Capsella bursa-pastoris (roots)

1. Roots were homogenized while blending with extraction mixture (1:4, w:v). Homogenate was filtered through a paper filter and centrifuged at 20,000g 30 min

2. The supernatant was concentrated using the reversed-phase C₁₈ Sep-Pack cartridge. Peptides were removed from the column by washing with 80% acetonitrile with 0.1% TFA

[6]

 Buffer solution: 0.1 M Tris–HCl (pH 7.2)

Defensin-like peptides

Phaseolus limensis (seeds)

1. Seeds were washed and soaked in water for 12 h. Then homogenated in a blender with buffer solution. The homogenate was centrifuged at 12,000 rpm for 20 min at 4 °C;

2. The supernatant was fractionated by two-step ammonium sulfate precipitation. In the first step, the solution was saturated to 20%; the resulting supernatant was saturated to 85%

3. After centrifugation at 12,000 rpm for 20 min, the precipitate was collected, dissolved in 100 mL of 0.01 M Tris–HCl buffer (pH 7.2), and dialyzed against the same buffer and subjected to the further separation

[43]

 Buffer solution: 10 mM Na₂HPO₄, 15 mM NaH₂PO₄, 100 mM KCl, 1,5% EDTA, pH 5.4

Thionin-like peptides

Capsicum anuum (Fruits without seeds)

1. C. anuum fruits were extracted with a buffer solution in a 1 to 5 ratio (w:v) for 2 h;

2. The extract was saturated with ammonium sulfate. The precipitate formed between 0 and 70% relative saturation was redissolved in distilled water and heated at 80 °C for 15 min and centrifuged;

3. The resulting suspension was extensively dialyzed against distilled water, freeze-dried and subjected to further fractionation by the chromatographic method

[18]

Organic solutions

 MeOH/CH₂Cl/0.05% TFA in water (4:4:1)

PawS-derived peptides

Zinnia haageana (seeds)

1. Seeds (50 mg) were ground to a fine powder with mortar and pestle under liquid nitrogen with a pinch of 0.1 mm glass beads;

2. The extraction mixture (0.9 mL) was added to the seed powder, and the mixture was vortexed and centrifuged (3 min at 16,000g). If the phases were not separated at this point, 0.1 mL of chloroform or 0.1 mL of 0.05% TFA in water was added alternately, followed by short centrifugation after each addition, until phase separation was achieved;

3. After phase separation, the upper polar layer was collected, and dried under vacuum and re-dissolved in 0.5 mL, 5% (v/v) formic acid for the further peptide identification

[44]

 EtOH or MeOH 20% or 50% in water respectively

Cyclotydes

Viola odorata (aerial parts)

1. Dried plant material was finely ground;

2. Plant material was extracted with an extraction mixture in a 1 to 20 (w:v) ratio for 6 h

[45]

 MeOH in water (1:1)

Thionins

Viscum album (Green and white parts)

1. Plant material was crushed, the extraction mixture was added (1:5, w:v), the solution was filtered and its volume was reduced;

2. The aqueous phase was successively partitioned with cyclohexane, dichloromethane, and ethyl acetate;

3. Ethanol was added to the concentrated aqueous phase to achieve 85% (v/v) concentration; the precipitate was separated by centrifuging (2000g; 10 min);

4. The supernatant was concentrated, and ethanol was added to 85% (v/v). The precipitates were pooled

[46]