Phloxine

Last updated
Phloxine [1]
Phloxine.png
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
ChemSpider
DrugBank
ECHA InfoCard 100.038.490 OOjs UI icon edit-ltr-progressive.svg
EC Number
  • 242-355-6
PubChem CID
UNII
  • InChI=1S/C20H4Br4Cl4O5.2Na/c21-5-1-3-17(9(23)15(5)29)32-18-4(2-6(22)16(30)10(18)24)20(3)8-7(19(31)33-20)11(25)13(27)14(28)12(8)26;;/h1-2,29-30H;;/q;2*+1/p-2
    Key: OOYIOIOOWUGAHD-UHFFFAOYSA-L
  • 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
AppearanceRed 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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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]

Contents

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 [ citation needed ]. 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

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. 1 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. 1 2 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. 1 2 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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