US20030157518A1 - Fluorescent compounds - Google Patents

Fluorescent compounds Download PDF

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US20030157518A1
US20030157518A1 US10/258,310 US25831003A US2003157518A1 US 20030157518 A1 US20030157518 A1 US 20030157518A1 US 25831003 A US25831003 A US 25831003A US 2003157518 A1 US2003157518 A1 US 2003157518A1
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ring
compound
double bond
organic compound
formula
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Philip Bell
Peter Karuso
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Fluorotechnics Pty Ltd
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Assigned to FLUOROTECHNICS PTY LIMITED reassignment FLUOROTECHNICS PTY LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BELL, PHILIP JOHN L., KARUSO, PETER
Publication of US20030157518A1 publication Critical patent/US20030157518A1/en
Priority to US11/949,525 priority Critical patent/US8114963B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B61/00—Dyes of natural origin prepared from natural sources, e.g. vegetable sources
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6827—Total protein determination, e.g. albumin in urine
    • G01N33/6839—Total protein determination, e.g. albumin in urine involving dyes, e.g. Coomassie blue, bromcresol green

Definitions

  • This invention relates generally to novel fluorescent compounds and to compounds which can function as fluorescent dyes, methods of isolating such compounds from microorganisms and uses of such compounds in scientific applications.
  • ligands are frequently used to confer a specificity for a biochemical reaction that is to be observed and the fluorescent dye provides the means of detection or quantification of the interaction.
  • these applications include, among others, the detection of proteins (for example in gels or aqueous solution), cell tracking, the detection of proteins via fluorescently labelled antibodies, the assessment of enzymatic activity, the staining of nucleic acids.
  • Red fluorescent compounds are used extensively in many fields of biological study. Many of these, including Texas red, Tetramethyl rhodamine-isothiocyanate or red emitting BODIPY dyes require excitation at green wavelengths such as 542 nm. This limits their use in many applications, especially those where the argon-ion laser is used for excitation.
  • Compounds such as ethidium bromide can be excited with light from the argon-ion laser (520 nm band), but are not generally suitable for tagging of organic molecules other than nucleic acids.
  • Other compounds such as phycoerythrin, can be excited using the argon-ion laser (488 nm) and does emit in the orange/red wavelengths.
  • Phycoerythrin however, has poor stability and a high molecular weight making it unsuitable for many applications such as cell tracking, labelling of nucleic acids or staining proteins.
  • fluorescein di- ⁇ -D-galactopyranoside is a non-fluorescent compound that is sequentially hydrolysed by the enzyme ⁇ -galactosidase first to generate fluorescein monogalactoside and then to fluorescein which is highly fluorescent.
  • the cleavage of the FDG compound can be monitored by the increase in fluorescence in the solution, and thus allows sensitive quantification of enzymatic activity.
  • fluorophores are suitable for this procedure. Therefore, novel fluorescent compounds that can be conjugated to a variety of substrates will be of utility.
  • the present inventors have isolated new compounds derived from a fungus that is capable of combining readily with a range of organic molecules to produce fluorescent complexes.
  • each of R, R′ and R′′ is a hydrogen atom, halogen atom or a straight or branched chain C 1-20 alkyl, alkenyl or alkynyl group optionally substituted with one or more halogen, hydroxy and/or oxy group;
  • rings A, B and C optionally include one or more double bonds
  • rings B and C are optionally substituted with one or more halogen atoms
  • the compound is capable of interacting with an organic compound and when interacting with the organic compound, the compound emits fluorescence after excitation at a broad range of wavelengths.
  • the compound is excited at wavelengths in the range of 300-560 nm, more preferably, 380-530 nm and even more preferably, UV wavelengths and/or blue wavelengths.
  • the compound emits in the wavelengths of 460 to 700 nm. More preferably its emission peak is centered around 530 nm when interacting with an organic compound such as sodium dodecyl sulphate and centered around 605 nm when interacting with a biomolecule such as a protein or cell.
  • the first aspect of the invention provides a compound according to formula (Ia):
  • the compound according to formula Ia has the stereochemistry as depicted in formula (Ib):
  • R is a straight or branched chain C 1-20 conjugated alkenyl group optionally substituted with hydroxy and oxy groups
  • R′ is a straight or branched chain C 1-20 alkyl group optionally substituted with a hydroxy group
  • R′′ is a straight or branched chain C 1-20 alkyl group.
  • R —C(OH)CHC(O)(CH) 6 CH 3
  • R′ —CH 2 OH
  • R′′ Me
  • the present invention according to the first and second aspects consists in the compound 5,6-dihydro-3-[(1Z, 4E, 6E, 8E)-1-hydroxy-3-oxo-1,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-g][2]benzopyran-2-9(9aH)-dione according to formula (Ic):
  • the present invention consists in a compound according to formula (I), wherein each of R, R′ and R′′ is a hydrogen atom, halogen atom or a straight or branched chain C 1-20 alkyl, alkenyl or alkynyl group optionally substituted with one or more halogen, hydroxy and/or oxy groups;
  • ring A, B and C are optionally substituted with one or more halogen atoms
  • rings A, B and C optionally include one or more double bonds; with the proviso that:
  • the compound according to second aspect of the invention is in accordance with formula (Ia) and more preferably, has the stereochemistry as depicted in formula (Ib).
  • R —C(OH)CHC(O)(CH) 6 CH 3
  • R′ —CH 2 OH
  • R′′ Me
  • the present invention according to the second aspect consists in the compound 5,6-dihydro-3-[(1Z, 4E, 6E, 8E)-1-hydroxy-3-oxo-1,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-g][2]benzopyran-2-9(9aH)-dione according to formula (Ic).
  • the compounds of formula (I)-(Ic), such as 5,6-dihydro-3-[(1Z, 4E, 6E, 8E)-1-hydroxy-3-oxo-1,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-g][2]benzopyran-2-9(9aH)-dione are members of the azaphilone compounds.
  • the azaphilone compounds are produced by a variety of fungi, and have been investigated for their antibiotic functions, their ability to inhibit enzymatic function, and their role as colouring agents in the food additives produced by Monascus sp.
  • the azaphilone nucleus has also been found in the pigments produced by Monascus sp.
  • the pigments containing the common azaphilone core include dihydro-monascin and monascorubin. There is no record of their utility as fluorescent dyes for the staining of biomolecules or organic compounds.
  • the present inventors have unexpectedly found that compounds according to formula (I)-(Ic) are not fluorescent in aqueous solution, but are capable of interacting with organic compounds to produce an intensely fluorescent stain.
  • the compounds according to formula (I)-(Ic) of the invention are used in the detection and tagging of organic molecules.
  • the compound according to formula (I)-(Ic), when bound to an organic molecule emits fluorescence after excitation at blue wavelengths. More preferably, the compounds according to formula (I)-(Ic) are excited by light in the absorbance range 300-560 nm.
  • a compound according to formula (I)-(Ic) interacts with proteins to produce a fluorescent complex that can be excited by light generated by standard UV transilluminators (307 nm). Upon excitation, the fluorescent complex emits light over a wide range of wavelengths allowing the protein complexes to be detected.
  • the excitation wavelength of the protein/dye complex includes that of ethidium bromide complexed with DNA (Absorption maximum 518 nm, Emission maxima 605 nm). This is of particular utility because it allows the same equipment to be used to detect both DNA and protein in gels.
  • the broad range of excitation wavelengths allows the compound to be excited strongly by 488 nm and 514 nm bands of the argon ion laser, as well as absorb light emitted from diode light sources such as those the emit at around 400 nm
  • a fluorescent form of the compound according to formula (I)-(Ic) is capable of strongly absorbing light from the 488 nm output of the argon-ion laser, and to re-emit the light at wavelengths longer than 600 nm.
  • the compound according to formula I is included in a composition, along with an analytically acceptable carrier, and may be used in combination with a fluorochrome such as fluorescein in dual staining applications.
  • a fluorochrome such as fluorescein in dual staining applications.
  • the compound according to formula (I)-(Ic) may be produced by a fungal species.
  • the fungal species is the strain deposited at the Australian Government Analytical Laboratories (AGAL) on Jan. 15, 1998 identified by Accession Number NM98/00507.
  • the binding of the compound according to formula (I)-(Ic) to organic molecules may involve direct chemical or physical binding or may be achieved by the use of “linking molecules” known to the art.
  • Organic molecules which may be bound by the compound of the invention when used as a fluorescent dye include, for example, proteins, peptides, sugars, nucleic acids, antibodies, cell surface biomolecules, detergents and cells. Due to the compound's fluorescent and organic compound-binding characteristics, it will be appreciated that the compound will have use in any application where detection of a fluorescent dye attached to an organic compound is required.
  • the present invention consists in a process of producing a compound according to the first and second aspect of the invention, the process comprising culturing a fungal species under conditions such that the fungal species produces the compound; and separating the compound from the culture.
  • the compound according to formula (I)-(Ic) can be used as a fluorescent dye when in a crude culture extract or when purified by extraction and separation techniques.
  • the compound according to formula I may also be produced synthetically by direct chemical synthesis, or by modification of intermediate(s) in the biosynthetic pathway used by the fungi.
  • the compound according to formula (I)-(Ic) of the present invention may also be produced synthetically by chemical means.
  • the knowledge that a new fluorescent compound is produced by fungi may lead to other means of producing the compound apart from culturing the fungi under the required conditions.
  • the compound according to formula (I)-(Ic) of the present invention has the distinct advantage that it binds to cells and other organic molecules in its fluorescent form so can be used as a means to track the cells or the other organic molecules when labelled with the compound.
  • the present invention consists in use of the compound according to the first and second aspect of the present invention as a fluorescent dye or marker, preferably in scientific techniques for staining, labelling and/or detecting organic molecules.
  • Examples of the use of the compound according to the first and second aspect of the present invention include but are not restricted to cell tracking dyes for microscopy, membrane fluidity dyes, conjugation with antibodies, conjugation to nucleic acids, cell surface ligand imaging dyes, conjugation to sugars, cytometric analysis, and confocal microscopy. It will be appreciated, however, that the compound would be suitable for any use where fluorescence in the red wavelengths is required, including when excited at 488 nm.
  • the present invention consists in a method of fluorescent-labelling an organic compound, the method comprising causing the compound according to the first or second aspect of the present invention to bind to an organic compound such that the organic compound is fluorescently labelled with the compound.
  • the fluorescently labelled organic compound is detected when exposed to a wide range of wavelengths, preferably wavelengths in the range of 300-560 nm.
  • the organic compound may include, but is not limited to, proteins, peptides, sugars, nucleic acids, antibodies, cell surface biomolecules, detergents and cells.
  • the compound may bind directly to the organic compound due to a chemical or physical association or may bind to the organic compound via a linking molecule. If the compound is attached to a ligand specific for the organic compound, for example an antibody or lectin, then the binding of that ligand to the organic compound will cause the organic compound to be fluorescently labelled.
  • the present invention consists in a method of detecting a organic compound in a sample including the organic compound, the method comprising labelling the organic compound according to the method of the fifth aspect of the present invention; and detecting the organic compound in the sample by monitoring or detecting its fluorescence.
  • the labelled organic compound is detected when the sample is exposed to light from a wide range of wavelengths, preferably wavelengths in the range of 300-560 nm, more preferably blue wavelengths.
  • the monitoring or detecting of the fluorescence of the labelled-organic compound may be by any means known to the art. Such means include, but not limited to, transillumination, spectroscopy microscopy and cytometry.
  • FIG. 1 shows an NMR spectrum of 5,6-dihydro-3-[(1Z, 4E, 6E, 8E)-1hydroxy-3-oxo-1,4,6,8-decatetraenyl]-6-hydroxymethyl-9a-methyl-2H-furo[3,2-g][2]benzopyran-2-9(9aH)-dione a fluorescent compound according to the first and second aspects of the invention.
  • FIG. 2 shows an example of an emission spectra of the compound identified by FIG. 1 when bound to bovine serum albumin (BSA) and propidium iodide bound to DNA.
  • BSA bovine serum albumin
  • a growth medium for the fungus AGAL Accession Number NM 98/00507, was prepared by adding 40 g Sucrose (CSR), 5 g Yeast Extract (Difco), 10 g Peptone (Difco) and 10 g Agar (Difco) to 1 L of water. The mixture was autoclaved for 15 min at 115° C. to both sterilise the medium and to dissolve the agar. The liquid was poured into culture dishes and allowed to set at room temperature. After cooling, a culture of the fungus was inoculated onto the surface of the medium, and incubated at 25° C. for three days. The culture was transferred to a refrigerator and incubated at 4° C. until the culture turned an intense red colour and dye production was high (usually 3 to 5 days).
  • the culture medium including the fungal biomass was transferred into ethanol at a ratio of one volume of culture medium to two volumes of ethanol.
  • the dye was extracted into the ethanolic phase by incubation at 4° C. and shaking for 16 hours.
  • the liquid phase was decanted from the residual culture medium, and centrifuged at 3000 rpm for 10 min at 4° C.
  • Extract B produced according to the method described in example 2 was subjected to HPLC purification (Supelco C16-amide column, resolved in 75% methanol/water, 0.05% acetic acid, then 50% acetonitrile/water, 0.05% acetic acid). The final fraction was frozen ( ⁇ 20° C.) to remove water and the remaining acetonitrile was evaporated under a stream of nitrogen.
  • the NMR sample was prepared by dissolving the HPLC purified compound obtained from Example 3 ( ⁇ 5 mg) in CDCl 3 (0.5 mL; 99.96 atom %, Aldrich) and filtering it into an NMR tube (PP527, Wilmald). The sample was degassed and equilibrated under an atmosphere of nitrogen.
  • the NMR data were acquired on a Bruker DRX600 (600 MHz) NMR spectrometer at 27° C. and processed using xwinNMR (version 2.6; Bruker). All 2D NMR experiments were run with quadrature detection with an 1 H spectral width of 6009 Hz and a recycle delay of 2 s. Chemical shifts were referenced to the residual CHCl 3 (dH 7.26 ppm; dC 77.01 ppm). High power 1 H p/2 pulses were determined to be 9.5 ms and low power (for MLEV spin lock) at 25.15 ms. 13 C high power p/2 pulse was 10.5 ms and a low power pulse of 65 ms was used for GARP decoupling. Gradient pulses were delivered along the z-axis using a 100 step sine program.
  • a 1000 step Gaussian program (60 ms, 64.6 dB) was used to achieve a p/2 pulse.
  • Gradient selection was achieved with a 15% gradient along the z-axis.
  • 10K transients were accumulated over 6009 Hz.
  • ROE enhancements were measured as a percentage of the irradiated peak and not compensated for offset from the carrier frequency.
  • Heteronuclear single quantum coherence was used to correlate and assign all protons to protonated carbons.
  • each spin system was characterised by running a series of total correlation spectroscopy (TOCSY) experiments with mixing times of 8 msec, 20 msec, 100 msec.
  • TOCSY total correlation spectroscopy
  • the shortest mixing time gave correlations to only the directly coupled protons whereas the longest mixing time identified the entire spin system and gave information about very small long range couplings which were valuable for assigning the positions of the many (apparently) uncoupled protons.
  • Thorough space connectivities were achieved by a series of one dimensional selective rotating frame correlation spectroscopy (ROESY) experiments.
  • Example 3 The compound produced according to Example 3 was subjected to high resolution mass spectrometry to determine that the molecular formula of the compound was C 23 H 22 O 7 .
  • a protein gel was prepared according to any one of a number of standard protocols.
  • an SDS page gel was prepared according to the protocol of Laemrnli (U.K. 1970, Nature, 227:680-685).
  • the gel was removed from the electrophoresis apparatus.
  • the gel was fixed in a container containing 100 mL of a staining solution composed of 90 mL water, 10 mL glycerol and 5 mL of Extract A.
  • the gel and staining solution were incubated with gentle shaking for 90 minutes. After the 90 minute staining procedure, the solution was removed and the gel briefly washed three times in water. After these brief washes, the gel was placed in 100 mL of 10% glycerol in water and incubated with shaking for a further 30 min.
  • the gel was transferred to a UV transilluminator (307 nm), and photographed using polaroid 667 black and white film and a filter set designed for the detection of ethidium bromide gels (eg. Molecular probes E-7591).
  • a micro-organism is cultured under conditions to promote sporulation and Extract B is used to facilitate the identification of sporulated cells within a predominantly vegetative cell culture.
  • Saccharomyces cerevisiae was grown in a water based broth containing 10 g/L Glucose, 5 g/L yeast extract, and 10 g/L peptone. After a 16 hour incubation at 30° C., the cells were harvested by centrifugation and resuspended in 1 mL of water. The 5 ⁇ L aliquots of the culture were spotted onto a semi-solid medium composed of 5% Potassium acetate, 2% Agar and water. The cultures were incubated at 22° C.
  • the sporulated culture was resuspended in water to a density of 1 ⁇ 10 9 cells per mL.
  • 5 ⁇ L of Extract B was added to 1 mL of the sporulated culture.
  • 5 ⁇ L of a 10 mM solution (in DMSO) of 5(6)-carboxyfluorecein diacetate (CFDA) was added to the sporulated culture.
  • the sporulated culture was harvested by centrifugation and resuspended in 1 mL of water. Examination under an epifluorescence microscope (excitation 488 nm) allowed for rapid differentiation between sporulated and vegetative cells.

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Electroluminescent Light Sources (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Plural Heterocyclic Compounds (AREA)
  • Luminescent Compositions (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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  • Cathode-Ray Tubes And Fluorescent Screens For Display (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
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US10/258,310 2000-04-26 2001-04-26 Fluorescent compounds Abandoned US20030157518A1 (en)

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US20070077202A1 (en) * 2005-06-12 2007-04-05 Pentax Corporation Histostain composition for endoscope

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US20030077263A1 (en) * 1999-10-29 2003-04-24 Immunex Corporation Method of activating dendritic cells
AUPQ711700A0 (en) 2000-04-26 2000-05-18 Macquarie Research Limited Fluorescent compounds
AU2003901361A0 (en) * 2003-03-25 2003-04-10 Fluorotechnics Pty Limited Method of enhancing fluorescence
WO2007051256A1 (en) * 2005-11-04 2007-05-10 Fluorotechnics Pty Limited Method for monitoring protein digestion
FR2951721A1 (fr) 2009-10-26 2011-04-29 Univ Rouen Procede de synthese d'analogues de l'epicocconone
CN110615777A (zh) * 2019-08-19 2019-12-27 中国科学院长春应用化学研究所 化合物及其制备方法和应用
CN111808116B (zh) * 2020-07-24 2023-04-07 西北农林科技大学 一种荧光类化合物及其制备方法与应用

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US5013565A (en) * 1990-08-03 1991-05-07 Uop Water-soluble red pigments from monascorubrin and rubropunctatin as food colorants
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AUPO470197A0 (en) * 1997-01-21 1997-02-13 Attfield, Paul Victor Fluorescent dyes from microorganisms
AUPQ711700A0 (en) 2000-04-26 2000-05-18 Macquarie Research Limited Fluorescent compounds
KR20030048473A (ko) 2000-11-17 2003-06-19 시바 스페셜티 케미칼스 홀딩 인크. 안료 형광성 증진 방법
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US3993789A (en) * 1974-11-07 1976-11-23 Societe D'assistance Technique Pour Produits Nestle S.A. Red pigment and process
US5013565A (en) * 1990-08-03 1991-05-07 Uop Water-soluble red pigments from monascorubrin and rubropunctatin as food colorants
US6046022A (en) * 1996-09-30 2000-04-04 Peking University Methods and compositions employing red rice fermentation products

Cited By (2)

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Publication number Priority date Publication date Assignee Title
US20070077202A1 (en) * 2005-06-12 2007-04-05 Pentax Corporation Histostain composition for endoscope
GB2433117B (en) * 2005-12-06 2011-03-09 Pentax Corp Histostain composition for an endoscope

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KR100855651B1 (ko) 2008-09-03
AUPQ711700A0 (en) 2000-05-18
KR20030016251A (ko) 2003-02-26
EP1309598A4 (en) 2004-04-14
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NZ522291A (en) 2004-10-29
EP1309598A1 (en) 2003-05-14
CA2407641A1 (en) 2001-11-01
HK1055742A1 (en) 2004-01-21
JP2003533446A (ja) 2003-11-11
DK1309598T3 (da) 2008-03-17
EP1309598B1 (en) 2007-10-31
CN1255409C (zh) 2006-05-10
US8114963B2 (en) 2012-02-14
WO2001081351A1 (en) 2001-11-01
DE60131176D1 (de) 2007-12-13

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