Natural fluorescent dye obtained from a marine invertebrate, compositions containing the said dye and their uses
AssigneeCouncil of Scientific and Industrial Research
InventorsUsha Goswami, Anutosh Ganguly
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Abstract
The present invention discloses the process of extraction, purification and characterization of a fluorescent dye from a marine echinoderm Holothuria scabra, compositions containing the dye and various applications of the dye.
Claims54
A bioactive extract obtained from a marine organism and useful as a natural fluorescent dye having the following characteristics:
- i. decolorization by a reducing agent,
- ii. not a synthetic compound,
- iii. crude extract of the dye is yellowish green in color,
- iv. purified dye being reddish brown colored powder when seen with the naked eye in the daylight,
- V. under tube light some hues of green are emitted,
- vi. amorphous in nature,
- vii. soluble in water,
- vili. insoluble in the organic solvents like ethanol, methanol and acetone,
- ix. is negatively charged,
- x. has a pH of 6.5,
- xi. presence of a phenolic group,
- xii. absence of a quinoid ring,
- xiii. absence of aromatic amine groups,
- xiv. non-proteinaceous in nature,
- xv. reducing sugar is absent,
- xvi. dye has nature of a biosurfactant,
- xvii. dye also showed antimicrobial qualities and when antimicrobial assay was performed, showed zone of inhibition,
- xviii. pigment cum dye is a fluorescent dye and emits fluorescence when excited with different wavelengths of UV and visible spectral ranges on a spectrophotometer,
- xix. UV, visible spectroscopy from 300 nm-700 nm and the peaks are marked at 379 nm and 439nm wavelengths,
- xx. UV, visible spectroscopy from 250 nm-350 nm and the peaks are at 272 nm and 299 nm wavelengths,
- xxi. fluorescent spectroscopy in the UV and visible spectra, when excited with UV 270 nm wavelength the fluorescence is emitted in the 324 nm-380 nm range which comes under the WVA wavelength range of ultraviolet rays of the sunlight,
- xxii. with excitation wavelength 450 nm in Fluorescent spectroscopy the fluorescence emission occurred at 500 nm-580 nm with maximum intensity,
- xxiii. with excitation wavelength 540 nm in Fluorescent spectroscopy, the fluorescence emission occurred at 500 mn-620 nm with maximum intensity,
- xxiv. with excitation wavelength 555 nm in Fluorescent spectroscopy, the fluorescence emission occurred at 575 nm-620 nm with maximum intensity,
- xxv. physical checking of Whatman Filter no. 1 dipped with dye concentration 1:40000 dilution under UV transilluminator and Gel Documentation system with UV bulbs of 260um-280 nm range emit bluish green hue color of fluorescence,
- xxvi. emits three different colored fluorescence at 3 different wavelengths of the UV and visible ranges of the fluorescent cubes of an epifluorescence microscope,
- xxvii. fluorescence blue color emission occur in the 380 nm-400 nm range of UVA when excited under ultra violet cube WU-330 nm-385 nm excitation range,
- xxviii. fluorescence yellow color emission occurs in the 500 nm-570 nm range when excited under WB cube of 450 nm-480 nm excitation range,
- xxix. fluorescence orange color emission occurs in the 570 nm-650 nm range when excited under WG cube of 510 nm-550 nm excitation range,
- xxx. the dye emits hues of grays under the ordinary transmitted light bulb of the epifluorescence microscope when seen under 10× objective,
- xxxi. the dye emitted these fluorescence colors even at a dilution range of 1:40000 times ( i.e. 1 gm powder of dye dissolved in 40 liters of ultrapure water),
- xxxii. the fluorescence of the extract persisted even after atleast lyear at the room temperature,
- xxxiii. the fluorescence of the dye is highly photostable and does not get deteriorated by long exposures to direct light, and
- xxxiv. the fluorescence of the dye does not change even when frozen at 20° C., a temperature at which the molecules are unable to attain the energy necessary for activation like in extracts from luminescent organisms.
A dye as claimed in claim I wherein the orange colored fluorescent emission is comparable to the orange fluorescence color of TRITC fluorochrome used as components of the nonradioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
A composition comprising a bioactive extract obtained from the marine sea- cucumber Holothuris scabra together with conventional additives and useful for the preparation of flexible polyvinyl chloride film that exhibits fluorescent colors.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives and useful in the preparation of coating compositions and inks.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives and useful in detection of leaks.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives and useful in undersea probes.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives and useful in photo chemotherapy of skin cancers.
A cosmetic composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives and useful as a fluorescent molecular probe in situ hybridization kits for molecular diagnostics.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives and useful as a component of non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry and molecular biology.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives and useful in immuno fluorescent detections.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives and useful as a counterstain of DIG-labeled oliogonucleotide probes and anti-DIG Fab-fragments.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives and useful in single and multiple cell quantitative fluorescence in single and multicolor flowcytometry applications.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra which can be used in experiments where various applications of fluorescent dyes are needed to be performed at field stations situated at subzero degree temperature areas.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives and useful as fluorochrome stains for epifluorescence microscopy.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives and useful for a quick check of biocontamination in the health food industry, cosmetic industry, pharmaceutical and chemical industries.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives and useful for rapid estimations of bio-contaminants in laboratory cultures.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives and useful for a rapid check of bio-pollutants under field conditions.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives and useful as a competitive inhibitor of cholinesterases.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives and useful in antimicrobial compositions.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives and useful as a biosurfactant in toiletry compositions.
A composition comprising a bioactive extract obtained from the marine seacucumber Holothuris scabra together with conventional additives and useful in pesticidal compositions.
A composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuris scabra together with conventional additives and useful as a natural colorant.
A bioactive composition containing an extract obtained from the marine sea-cucumber Holothuris scabra in the ratio of 1:40000 in ultrapure water to obtain fluorescences of three colors at three different wavelengths.
A bioactive composition containing an extract obtained from the marine seacucumber Holothuris scabra to obtain a phase contrast and histochemical counterstain effect for different biochemical constituents of cells under transmitted light.
A skin care composition containing the dye together with physiologically and cosmetically acceptable vehicle such as diluent, dispersant or carrier.
A process for extraction of a natural fluorescent dye from Holothuris scabra sea cucumber, which comprises the steps of:
- a) collecting the material from seashore, changing of seawater and maintenance in the laboratory tanks without any mechanical aeration overnight,
- b) freezing the animals at −20° C.,
- c) thawing the animal to obtain the pigment;
- and
- d) repeating the steps (b) and (c) 3-4 times for removal of pigment and, if desired, purify the pigments.
A process for extraction of a natural fluorescent dye from Holothuris scabra sea cucumber which comprises the steps of extraction of the pigment from the skin of the sea cucumber Holothuris scabra by osmotic shocks preferably with adding 50% ethanol to the ultrapure water on first time, and repeating this at least 4 times to obtain the dye.
Dependencies are inferred from the claim text.