Phloxine

Phloxine B (commonly known simply as phloxine) is a water-soluble red dye used for coloring drugs and cosmetics in the United States[2] and coloring food in Japan.[3] It is derived from fluorescein, but differs by the presence of four bromine atoms at positions 2, 4, 5 and 7 of the xanthene ring and four chlorine atoms in the carboxyphenyl ring.[4] It has an absorption maximum around 540 nm and an emission maximum around 564 nm.[5] Apart from industrial use, phloxine B has functions as an antimicrobial substance, viability dye and biological stain.[6] For example, it is used in hematoxylin-phloxine-saffron (HPS) staining to color the cytoplasm and connective tissue in shades of red.[7]

Phloxine[1]
Names
Preferred IUPAC name
Disodium 2′,4′,5′,7′-tetrabromo-4,5,6,7-tetrachloro-3-oxo-3H-spiro[[2]benzofuran-1,9′-xanthene]-3′,6′-bis(olate)
Other names
Cyanosin; Cyanosine; Eosine bluish; Eosine Blue; Cyanosin B; Eosin Blue; Phloxine P; Phloxin B; Eosine I Bluish; Acid red 92; C.I. 45410; D & C Red no. 28
Identifiers
3D model (JSmol)
ChEBI
ECHA InfoCard 100.038.490
UNII
  • C1=C2C(=C(C(=C1Br)[O-])Br)OC3=C(C(=C(C=C3C24C5=C(C(=C(C(=C5Cl)Cl)Cl)Cl)C(=O)O4)Br)[O-])Br.[Na+].[Na+]
Properties
C20H2Br4Cl4Na2O5
Molar mass 829.63 g·mol−1
Appearance Red to brown powder
Soluble
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
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Infobox references

Antimicrobial properties

Lethal dosage levels

In the presence of light, phloxine B has a bactericidal effect on gram-positive strains, such as Bacillus subtilis, Bacillus cereus, and several methicillin-resistant Staphylococcus aureus (MRSA) strains.[8] At a minimum inhibitory concentration of 25 μM, growth is reduced by 10-fold within 2.5 hours. At concentrations of 50 μM and 100 μM, growth is stopped completely and cell counts decrease by a factor of 104 to 105.[6] For humans, the Food and Drug Administration deems phloxine B to be safe up to a daily dosage of 1.25 mg/kg.[2]

Mechanism of action

Bacteria exposed to phloxine B die from oxidative damage. Phloxine B ionizes in water to become a negatively charged ion that binds to positively charged cellular components . When phloxine B is subjected to light, debromination occurs and free radicals and singlet oxygen are formed. These compounds cause irreversible damage to the bacteria, leading to growth arrest and cell death.[8] Gram-negative bacteria are phloxine B-resistant due to the outer cell membrane that surrounds them. This polysaccharide-coated lipid bilayer creates a permeability barrier that prevents efficient uptake of the compound. Addition of EDTA, which is known to strip the lipopolysaccharides and increase membrane permeability,[9] removes the phloxine B resistance and allows gram-negative bacteria to be killed as well.

Measure of viability
Further information: Viability assay

Phloxine B can be used to stain dead cells of several yeasts, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. When diluted in yeast growth media, the dye is unable to entere cell because of their membranes. Dead yeast cells lose membrane integrity, so phloxine B can enter and stain the intracellular cytosolic compounds. Therefore, staining is a measure of cell death. In cell counting assays, the number of fluorescent (i.e. dead) cells observed through a haemocytometer can be compared to the total number of cells to give a measure of mortality.[10] The same principle can be applied at higher throughput by fluorescence-activated flow cytometry (FACS), where all phloxine B-stained cells in a sample are counted.[11] [Note: some reports suggest that phloxine B is instead pumped out of live yeast cells but retained in dead/dying yeast cells.[12][13] However, definitive evidence for either model is still needed.]

References
  1. Phloxine B (Acid red 92)
  2. Food and Drug Administration (2001). The Code of Federal Regulations of the United States of America, Title 21, Part 74.1328. U S Government Printing Office. p. 296. Retrieved 15 April 2016.
  3. Kamikura, M (1970). "Thin Layer Chromatography of Synthetic Dyes (X)". Food Hygiene and Safety Science (Shokuhin Eiseigaku Zasshi). 11 (4): 242–248. doi:10.3358/shokueishi.11.242.
  4. Duarte, Paulo; Ferreira, Diana P.; Ferreira Machado, Isabel; Filipe, Luis; Ferreira, Vieira; Rodríguez, Hernan B.; San Román, Enrique (2012). "Phloxine B as a Probe for Entrapment in Microcrystalline Cellulose". Molecules. 17 (2): 1602–1616. doi:10.3390/molecules17021602. PMC 6268435. PMID 22314381.
  5. Coppeta, J.; Rogers, C. (1998). "Dual emission laser induced fluorescence for direct planar scalar behavior measurements". Experiments in Fluids. 25 (1): 1–15. Bibcode:1998ExFl...25....1C. doi:10.1007/s003480050202. S2CID 37649159.
  6. Rasooly, Avraham; Weisz, Adrian (2002). "In Vitro Antibacterial Activities of Phloxine B and Other Halogenated Fluoresceins against Methicillin-Resistant Staphylococcus aureus". Antimicrobial Agents and Chemotherapy. 46 (11): 3650–3653. doi:10.1128/AAC.46.11.3650-3653.2002. PMC 128710. PMID 12384384.
  7. Borgerink, Hermina. "HPS stain". Narkive Mailing List Archive. Retrieved 18 April 2016.
  8. Rasooly, Reuven (August 2005). "Expanding the bactericidal action of the food color additive phloxine B to gram-negative bacteria". FEMS Immunology & Medical Microbiology. 45 (2): 239–244. doi:10.1016/j.femsim.2005.04.004. PMID 15949926.
  9. Leive, Loretta; Kollin, Virginia (July 1967). "Controlling EDTA treatment to produce permeable escherichia coli with normal metabolic processes". Biochemical and Biophysical Research Communications. 28 (2): 229–236. doi:10.1016/0006-291x(67)90434-2. PMID 4166571.
  10. Noda, Takeshi (2008). "Chapter 2 Viability Assays to Monitor Yeast Autophagy". Autophagy: Lower Eukaryotes and Non-Mammalian Systems, Part A. Methods in Enzymology. Vol. 451. pp. 27–32. doi:10.1016/S0076-6879(08)03202-3. ISBN 9780123745484. PMID 19185710.
  11. Guérin, Renée; Beauregard, Pascale B.; Leroux, Alexandre; Rokeach, Luis A. (16 July 2009). "Calnexin Regulates Apoptosis Induced by Inositol Starvation in Fission Yeast". PLOS ONE. 4 (7): e6244. Bibcode:2009PLoSO...4.6244G. doi:10.1371/journal.pone.0006244. PMC 2705804. PMID 19606215.
  12. Kwolek-Mirek, Magdalena; Zadrag-Tecza, Renata (September 2014). "Comparison of methods used for assessing the viability and vitality of yeast cells". FEMS Yeast Research. 14 (7): 1068–1079. doi:10.1111/1567-1364.12202. PMID 25154541.
  13. Minois, Nadège; Frajnt, Magdalena; Wilson, Chris; Vaupel, James W. (11 January 2005). "Advances in measuring lifespan in the yeast Saccharomyces cerevisiae". Proceedings of the National Academy of Sciences of the United States of America. 102 (2): 402–406. Bibcode:2005PNAS..102..402M. doi:10.1073/pnas.0408332102. PMC 544282. PMID 15625107.
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