Bacterial plasma membrane. Gramicidin assembles as transmembrane channels consisting of two molecules associated head-to-head, forming a β-helix structure with an internal pore that allows permeation of cations across the membrane, disrupting ionic balance. No specific IC₅₀ or Kᵢ values were reported in this study. [1]
Gramicidin targets bacterial cell membranes by forming ion channels that allow the passage of monovalent cations (particularly K⁺ and Na⁺) across the lipid bilayer. This disrupts the electrochemical gradient across the bacterial membrane, leading to loss of cellular ions, dissipation of membrane potential, and ultimately bacterial cell death. Gramicidin primarily affects Gram-positive bacteria due to its inability to penetrate the outer membrane of Gram-negative bacteria.

Antimicrobial activity against bacteria: Gramicidin alone showed poor activity against Escherichia coli (Gram-negative) with a minimum bactericidal concentration (MBC) > 20 μM, but was effective against Staphylococcus aureus (Gram-positive) with an MBC of 4.0 μM. [1]
Cytotoxicity against eukaryotic cells: Gramicidin alone was highly toxic to Saccharomyces cerevisiae (a eukaryotic yeast model), with significant cell death observed at concentrations as low as 1 μM. [1]
Mechanism of action: Gramicidin forms functional ion channels in dioctadecyldimethylammonium bromide (DODAB) lipid bilayers, as demonstrated by osmotic swelling assays. Addition of hyperosmotic KCl or glucose to DODAB large vesicles (LV) containing Gr caused vesicle swelling rather than shrinkage, indicating increased cation permeability. Leakage of phosphorylated compounds from bacteria treated with DODAB/Gr formulations confirmed bacterial cell lysis. [1]
Spectroscopic characterization: The intrinsic fluorescence of Gr (due to tryptophan residues) and circular dichroism (CD) spectra were used to characterize Gr conformation in different environments. In DODAB LV, Gr exhibited a CD spectrum resembling that in 2,2,2-trifluoroethanol (TFE), indicating a β-helix conformation. In DODAB bilayer disks (BF), Gr displayed a CD spectrum more similar to that in ethanol, suggesting different localization (adsorbed at BF borders rather than inserted). [1]
Gramicidin is an antimicrobial peptide that forms membrane channels and raises the permeability of those channels to cations. In contrast to its action on Staphylococcus aureus, gramicidin exhibits weak effectiveness against Escherichia coli and Saccharomyces cerevisiae, as evidenced by the survival rate (%) of bacteria within a specific dose range. Saccharomyces cerevisiae is not harmed by bactericidal activity, which is observed over a range of low gramicidin and DODAB concentrations [1].
In vitro, gramicidin demonstrates potent antibacterial activity against Gram-positive bacteria including Staphylococcus aureus, Streptococcus species, and other susceptible organisms. Minimum inhibitory concentrations (MICs) are typically in the low microgram per milliliter range. The compound's ion channel-forming activity can be studied using artificial lipid bilayers or liposome assays where gramicidin-induced ion flux is measured. Gramicidin also exhibits hemolytic activity against red blood cells due to its membrane-disrupting properties.

In vivo, gramicidin is used topically for the treatment of skin infections, wound infections, and superficial bacterial infections. It is typically formulated in combination with other antibiotics (neomycin and polymyxin B) to provide broad-spectrum coverage. Gramicidin is not administered systemically due to its toxicity, including hemolysis and nephrotoxicity. Topical application results in localized antibacterial activity with minimal systemic absorption.

Antibacterial susceptibility testing: Gram-positive bacterial strains are cultured in appropriate media and treated with gramicidin at various concentrations (typically 0.1-100 µg/mL). MICs are determined by broth microdilution or agar dilution methods according to CLSI guidelines. For ion channel studies, gramicidin is incorporated into artificial lipid bilayers and single-channel currents are measured using patch-clamp or planar bilayer electrophysiology.

Microbial viability assay (CFU counting): E. coli, S. aureus, or S. cerevisiae cells were mixed with various concentrations of gramicidin (alone or incorporated into DODAB formulations) in 0.264 M D-glucose solution and incubated for 1 hour. Serial dilutions were plated on agar plates, incubated for 24–48 hours, and colony-forming units (CFU) were counted. Cell survival (%) was calculated relative to controls without antimicrobial agents. [1]
Bacterial cell lysis assay (inorganic phosphorus release): Bacteria were pelleted and resuspended in gramicidin dispersions in 1 mM NaCl. After 1 hour of interaction, the supernatant was collected and inorganic phosphorus concentration was determined. Bacterial lysis was expressed as percent leakage relative to control (bacteria lysed by sonication or other means). [1]
Scanning electron microscopy (SEM): Bacteria treated with DODAB/Gr formulations above the MBC were fixed, dehydrated, and sputter-coated with gold before imaging. SEM revealed distorted cell morphology, including cell enlargement and surface alterations. [1]
Osmotic swelling assay: DODAB large vesicles (LV) with or without incorporated gramicidin were prepared. Addition of hyperosmotic KCl or glucose solutions to the dispersion was followed by monitoring turbidity at 400 nm over time. Increased turbidity indicated vesicle shrinkage, while decreased turbidity indicated swelling. Gr-containing LV showed swelling upon hyperosmotic challenge, confirming functional channel formation. [1]
Fluorescence spectroscopy: Gr intrinsic fluorescence was measured at λₑₓc = 280 nm. The emission spectra were recorded for Gr in TFE, ethanol, DODAB LV, and DODAB BF to assess the microenvironment of tryptophan residues. [1]
Circular dichroism (CD) spectroscopy: CD spectra of Gr were acquired in the 200–280 nm range in different media (TFE, ethanol, DODAB LV, DODAB BF) to determine Gr secondary structure and conformation. [1]
Dynamic light scattering (DLS) and zeta potential: Hydrodynamic diameter, polydispersity index, and zeta potential of DODAB/Gr dispersions were measured at 90° scattering angle to characterize particle size and surface charge. [1]
For antibacterial assays, bacterial strains (e.g., S. aureus, S. epidermidis) are cultured in Mueller-Hinton broth or agar at 37°C. Gramicidin is serially diluted and added to bacterial cultures. After overnight incubation, the lowest concentration that inhibits visible bacterial growth is recorded as the MIC. For hemolysis assays, red blood cells are incubated with gramicidin at various concentrations and hemoglobin release is measured spectrophotometrically.

In vivo studies of gramicidin are primarily conducted in topical infection models. Animal models of skin infection or wound infection are established and gramicidin-containing formulations are applied topically. Efficacy is assessed by measuring bacterial load in infected tissues, wound healing rates, and clinical signs of infection. Systemic toxicity studies have been conducted to establish the safety profile for topical use.

Gramicidin has a molecular weight of approximately 1882.3 (for gramicidin A) and formula C₉₉H₁₄₀N₂₀O₁₇. It is a mixture of three related compounds (gramicidin A, B, and C) with similar structures. The compound is soluble in alcohol and other organic solvents but has limited water solubility. It is typically stored as a powder at -20°C or 4°C and protected from light and moisture.

Cytotoxicity against eukaryotic cells: Gramicidin alone was highly toxic to S. cerevisiae, with cell viability significantly reduced at concentrations above 1 μM. However, when incorporated into DODAB formulations (DODAB BF/Gr or DODAB LV/Gr), the toxicity was substantially reduced, with yeast viability remaining high at bactericidal concentrations. [1]
Minimum bactericidal concentrations (MBC): The MBC of gramicidin alone against S. aureus was 4.0 μM. In combination with DODAB, the MBC for Gr decreased to 0.80 μM (with DODAB LV) and 0.32 μM (with DODAB BF), indicating improved delivery and reduced effective dose. [1]

[1]. Novel gramicidin formulations in cationic lipid as broad-spectrum microbicidal agents. Int J Nanomedicine. 2014 Jun 30;9:3183-92.

There have been reports and data regarding the presence of gramicidin in Brevibacillus brevis. Gramicidin is a heterogeneous mixture of six antibiotic peptides extracted from the soil bacterium Bacillus brevis. Gramicidin is effective against most Gram-positive bacteria and some Gram-negative bacteria. It is administered topically. This is a group of peptide antibiotics derived from Bacillus brevis. Gramidin C or S is a cyclic ten-amino acid polypeptide, while Gramidin A, B, and D are linear. Gramicidin is one of the two main components of tyrosinin.
Gramicidin is a polypeptide antibiotic that forms ion channels in bacterial cell membranes, disrupting the electrochemical gradient and leading to bacterial cell death. It is primarily used topically in combination with other antibiotics for the treatment of skin infections and wound infections. Gramicidin is not effective against Gram-negative bacteria and is not used systemically due to its hemolytic toxicity. It has been used in clinical practice for many years as a topical antibacterial agent.

C99H140N20O17

1882.29472351074

1810.033419

1405-97-6

16130140

N-formyl-L-valyl-glycyl-D-leucyl-L-alanyl-D-valyl-L-valyl-D-valyl-L-tryptophyl-D-leucyl-L-tryptophyl-D-leucyl-L-tryptophyl-D-leucyl-L-tryptophyl-glycinol; For-Val-Gly-D-Leu-Ala-D-Val-Val-D-Val-Trp-D-Leu-Trp-D-Leu-Trp-D-Leu-Trp-Gly-ol

VGLAVVVWLWLWLWG

White to off-white solid powder

229-230℃

0mmHg at 25°C

10.9

21

17

52

136

3980

14

O=C([C@@H](CC(C)C)NC([C@H](CC1=CNC2C=CC=CC1=2)NC([C@H](C(C)C)NC([C@H](C(C)C)NC([C@@H](C(C)C)NC([C@H](C)NC([C@@H](CC(C)C)NC([C@H](C)NC(CNC([C@H](C(C)C)NC=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)N[C@H](C(N[C@@H](C(N[C@H](C(N[C@@H](C(N[C@H](C(NCCO)=O)CC1=CNC2C=CC=CC1=2)=O)CC(C)C)=O)CC1=CNC2C=CC=CC1=2)=O)CC(C)C)=O)CC1=CNC2C=CC=CC1=2

ZWCXYZRRTRDGQE-SORVKSEFSA-N

InChI=1S/C99H140N20O17/c1-51(2)37-73(109-86(123)59(17)107-81(122)49-105-96(133)82(55(9)10)106-50-121)89(126)108-60(18)87(124)117-84(57(13)14)98(135)119-85(58(15)16)99(136)118-83(56(11)12)97(134)116-80(44-64-48-104-72-34-26-22-30-68(64)72)95(132)112-76(40-54(7)8)92(129)115-79(43-63-47-103-71-33-25-21-29-67(63)71)94(131)111-75(39-53(5)6)91(128)114-78(42-62-46-102-70-32-24-20-28-66(62)70)93(130)110-74(38-52(3)4)90(127)113-77(88(125)100-35-36-120)41-61-45-101-69-31-23-19-27-65(61)69/h19-34,45-48,50-60,73-80,82-85,101-104,120H,35-44,49H2,1-18H3,(H,100,125)(H,105,133)(H,106,121)(H,107,122)(H,108,126)(H,109,123)(H,110,130)(H,111,131)(H,112,132)(H,113,127)(H,114,128)(H,115,129)(H,116,134)(H,117,124)(H,118,136)(H,119,135)/t59-,60-,73+,74+,75+,76+,77-,78-,79-,80-,82-,83-,84+,85-/m0/s1

(2R)-2-[[(2S)-2-[[2-[[(2S)-2-formamido-3-methylbutanoyl]amino]acetyl]amino]propanoyl]amino]-N-[(2S)-1-[[(2R)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2R)-1-[[(2S)-1-[[(2R)-1-[[(2S)-1-[[(2R)-1-[[(2S)-1-(2-hydroxyethylamino)-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-1-oxopropan-2-yl]-4-methylpentanamide

Gramicidine; Bacillus brevis gramicidin D; 1393-88-0; ...; 1405-97-6;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~53.13 mM)
H2O : ~1 mg/mL (~0.53 mM)

Solubility in Formulation 1: 10 mg/mL (5.31 mM) = in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 10 mg/mL (5.31 mM) in 5% DMSO + 95% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.=

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Solubility in Formulation 3: ≥ 2.5 mg/mL (1.33 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.5 mg/mL (1.33 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)