Claims
- 1. A bioactive semi-purified extract containing non-toxic proteinaceous fluorescent dye obtained from female gonads i.e. ovarian tissues of marine organism Holothuria scabra, occurring in intertidal, submerged, shallow and deep waters, usually abundant in shaded areas such as alcoves, crevices, ledges, overhangings, rocky sandy habitats, having dull to bright colored with or without exo- and endo skeleton, sessile, sedentary drifters, nektonic with varied swimming internal power usually nocturnal in habit, liabile to active predation, with and without luminescent, having external fertilization of eggs and fluorescent pigments giving emissions in few to all wavelength ranges of UTVB, UVA, visible colored spectrums and infra red spectrum.
- 2. A dye as claimed in claim 1 is useful as a natural fluorescent and having the following characteristics:
1. is a non-reduable dye, 2. not a synthetic compound but a natural compound, 3. crude extract of the dye is yellowish orange in color, 4. partially purified dye is light orangish in color when seen with the naked eye in daylight, 5. emits hues of multicolors under tube light, 6. exists in liquid form, 7. soluble in 70% ethyl alcohol, 8. 70% alcohol solution can be further diluted with water for use in experiments, 9. is negatively charged, 10. pH of the alcoholic solution is in the range of 6.8-7.5, 11. reducablle fluorophore is absent, 12. dye solution is proteinaceous in nature, 13. dye is nontoxic to bacteria E.coli, 14. dye is nontoxic to eukaryotic cells like oyster eggs and sperms, 15. dye is cell membrane permeant, 16. permeable to the nuclear membrane, 17. stains cliromatin, 18. TLC of dye solution shows four compounds having Rf values 0.917, 0.818, 0.661 and 0.463, 19. retention time of proteins in HPLC with UV detector at 280 nm shows 7 peaks, 20. retention time of peak 1 is 2.1 and its area is 116103, 21. retention time of peak 2 is 2.5 and its area is 38205, 22. retention time of peak 3 is 3.7 and its area is 7332, 23. retention time of peak 4 is 6.1 and its area is 31924, 24. retention time of peak 5 is 6.8 and its area is 535, 25. retention time of peak 6 is 7.6 and its area is 3684, 26. retention time of peak 7 is 8.8 and its area is 89653, 27. retention time of peptides by BPLC by UV detector at 205 nm has 10 peaks, 28. it has carbohydrate, 29. it has a glycoprotein, 30. it is inducing agglutinating lectin like qualities, 31. it agglutinates bacteria E.coli, 32. the agglutinated clumps do not die and they show fluorescence, 33. the dye agglutinates sperms to the egg membrane of oysters, 34. it facilitates sperm insemination and enhances rate of fertilization of oyster eggs, 35. the dye solution is stored at 4 degree centigrade, 36. once it attaches to the cell membranes, it stays fluorescent for several days, 37. it does not get photobleached once stained on cells, 38. dye does not stain dead cells of prokaryotes, 39. dye does not stain dead cells of oysters, 40. 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, 41. pigment cum dye is a fluorescent dye, which emits fluorescence when excited with different wavelengths of UV and visible spectral ranges on a spectrophotometer, 42. UV, visible spectroscopy the wavelength of excitation was maximum at 351 nm, 580 nm, 720 nm and the fluorescence emission spectrometric analysis was in between 400 to 600 nm when excited at 351 nm, 43. the emission maximas have two peaks at 450 nm and 550 nm, 44. physical checking of Whatman Filter no. 1 dipped with dye concentration 1:20000 dilution under Uw transilluminator and Gel Documentation system with UV bulbs of 260 nm-280 nm range emit bluish green hue color of fluorescence, 45. dye emits three different colored fluorescence at 3 different wavelengths of the UV and visible ranges of the fluorescent cubes of an epifluorescence nicroscope, 46. dye when attached to a cell membrane emits three different colored fluorescence at 3 different wavelengths of the UV and visible ranges of the fluorescent cubes of an epifluorescence microscope which are different from that of the dye alone, 47. on excitation of dye solution under UV cube in the range of 330 nm-385 nm, fluorescence of blue color emission occurs between 450 nm and 470 nm, 48. on excitation of dye solution under WB cube in the range of 450 nm-480 nm, fluorescence of green color emission occurs between 510 nm and 570 nm, 49. on excitation of dye solution under WG cube in the range of 510 nm-550 nm, fluorescence of orange color emission occurs between 610 nm and 650 nm, 50. the dye emits hues of yellowish grays under the ordinary transmitted light bulb of the epifluorescence microscope when seen under 100× objective, 51. on excitation of dye attached to a cell membrane under UV cube WU in the range of 330-385 nm, fluorescence of blue color emission occur between 470 nm-500 nm, 52. on excitation of dye attached to a cell membrane under WB cube of 450 nm -480 nm excitation range, fluorescence of green color emission occurs between 570 nm-610 nm, 53. on excitation of dye attached to a cell membrane under WG cube of 510 nm -550 nm excitation range, fluorescence of orange color emission occurs between 610 nm-650 nm, 54. the dye attached to a cell membranes emits hues of yellowish grays under the ordinary transmitted light bulb of the epifluorescence microscope when seen under 100× objective, 55. the dye emits fluorescence colors even at a dilution range of 1:400000 times, and 56. the fluorescence of the dye is highly photostable after staining the cell membranes and does not get deteriorated by long exposures to direct light, and the fluorescence of the dye does not change even when frozen at minus 20° C., a temperature at which the molecules are unable to attain the energy necessary for activation like in extracts from luminescent organisms.
- 3. The dye as claimed in claim 1 wherein, the multicolored emissions of the dye at different wavelengths of excitations are comparable to the fluorochrome microscopic stains already in the market.
- 4. The dye as claimed in claim 1 wherein, the blue colored fluorescence of the present dye is comparable to the emission of same color by DAPI fluorochrome at the same wavelength excitation, used as components of the non-radioactive labeling kits of biochemistry, cell biology, immunochemistry, and molecular biology.
- 5. The dye as claimed in claim 1 wherein the yellow colored fluorescence of the said dye in the visible range is comparable to the same colored emissions of Auramin used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
- 6. A dye as claimed in claim 1 wherein the yellow colored fluorescence of the said dye in the visible range is comparable to the same colored emissions of FITC used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
- 7. The dye as claimed in claim I wherein the orange colored fluorescent emission is comparable to the orange fluorescence color of Propidium Iodide fluorochrome used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
- 8. The dye as claimed in claim 1 wherein the orange colored fluorescent emission is comparable to the orange fluorescence color of Rhodamine fluorochrome used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
- 9. The dye as claimed in claim 1 wherein the orange colored fluorescent emission is comparable to the orange fluorescence color of TRITC fluorochrome used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
- 10. The dye as claimed in claim 1 wherein the dye is stable at the room temperature and has a long shelf life.
- 11. The dye as claimed in claim 1 wherein the molecular and radioactive kits of the said dye can be exported at the room temperatures.
- 12. The dye as claimed in claim 1 wherein the single dye has characteristics of at least one hundred different fluorochromes namely DAPI, Hoechest 33258, Hoechest 33342, FITC, acridine orange, auramine, Rhodamine, TRITC, and propidium iodide, etc, which are now in the market.
- 13. The dye as claimed in claim 1 wherein it can be used in all applications where presently Plhycobiliproteins are used as unlike them the dye does not undergo loss in fluorescence upon freezing.
- 14. The dye as claimed in claim 1 wherein under bright field of fluorescent microscope when seen under 10 × objective the hues of bluish grays produce a phase contrast effect which is useful in rapid screening of cytogentical, cytological, and histochemical slides and save expenses on the extra phase contrast accessory component of microscope.
- 15. The dye as claimed in claim 1 wherein under 100× oil immersion objective of an ordinary transmitted light microscope the proteins of yolk, nucleoplasm and chromatin of actively dividing cleavage cells show different colors of staining in the hues of brownish yellow for former, yellow for the latter and dark blue for the last cell component. This can be useful in rapid bioassays of effect can be seen on the various histochemical components of the cells.
- 16. The dye as claimed in claim 1 wherein the fluorescence color emissions follow Stoke's law of fluorescence.
- 17. The dye as claimed in claim 1 wherein the microphotographs with Kodak film rolls show hues of the adjacent color emission wavelengths such as blue color fluorescence under the epifluorescence.
- 18. The dye as claimed in claim 1 wherein the microphotographs with Kodak film rolls shows hues of the adjacent color emission wavelengths like when seen yellow color fluorescence under the epifluorescence microscope in microphotograph the hues of green also comes.
- 19. The dye as claimed in claim 1 wherein the orange fluorescence color seen under the epifluorescence microscope in microphotograph, the hues of red also comes.
- 20. The dye as claimed in claim 1 wherein the cytogenetic slides seen under all fluorescences gives a counterstain effect of cells and cell components versus the background color where no specimen but only dye is present.
- 21. The dye as claimed in claim 1 wherein, the dye solution diluted with water in the ratio above 1:400,000 times gives fluorescence of six colors at different wavelengths.
- 22. The dye as claimed in claim 1 wherein the dye solution diluted in the ratio of 1:400,000 and above gives fluorescence of six colors at three different wavelengths irrespective of partially purified dye solution or the stained cells.
- 23. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives useful in the preparation of coating compositions and inks.
- 24. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives useful in detection of leaks.
- 25. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives useful in undersea probes.
- 26. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives useful as a fluorescent molecular probe in situ hybridization kits for molecular diagnostics.
- 27. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives useful as a component of non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry and molecular biology.
- 28. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives useful in immuno fluorescent detections.
- 29. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives useful as a counterstain of DIG-labeled oliogonucleotide probes and anti-DIG Fab-fragments.
- 30. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives useful in single and multiple cell quantitative fluorescence in single and multicolor flowcytometry applications.
- 31. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives useful for conducting experiments at field stations situated at subzero degree temperature area.
- 32. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives useful as fluorochrome stains for epifluorescence microscopy.
- 33. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives useful for a quick check of biocontamination in the health food industry, cosmetic industry, pharmaceutical and chemical industries.
- 34. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives useful for rapid estimations of bio-contaminants in laboratory cultures.
- 35. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives useful for a rapid check of bio-pollutants under field conditions.
- 36. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives useful as a competitive inhibitor of cholinesterases.
- 37. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives useful in cell permeant dye compositions.
- 38. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives useful as a fertility enhancer.
- 39. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives in the ratio of 1:20000 to obtain fluorescence of three colors at three different wavelengths.
- 40. A fluorescent composition comprising bioactive extract as claimed in claim 1, together with suitable additives to obtain a phase contrast and histochemical counterstain effect for different biochemical constituents of cells under transmitted light.
- 41. A skin care composition comprising bio active extract together with physiologically and cosmetically acceptable vehicle such as diluent, dispersant or carrier.
- 42. A bioactive extract containing a fluorescent dye obtained from ovarian tissues of marine organism is used for the following applications:
1. use of fluorescent colors in variety of paints, inks, textiles; 2. a composition of fluorescent dye for bleaching and brightening polymer; 3. leak detection with a full spectrum fluorescent dye; 4. use in automated chemical metering system; 5. to mark location of crashed air-crafts, life crafts, and equipment for example rockets; 6. under sea probes; 7. chromatophore sunscreen component of cosmetics creams and lotions; 8. fluorescent in situ hybridization application kit component for molecular diagnostics; 9. component of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology for labeling of DNA, RNA, proteins and enzymes; 10. immunolfluorescent detection's; 11. counterstain of DIG-labeled oligonucleotide probes and Anti-DIG Fab-fragments and single and multiple flow cytometry applications; 12. fluorochrome stains for epifluorescence microscopy; 13. for a quick check of biocontamination in the health food industry, cosmetic industry, pharmaceutical and chemical industries; 14. for rapid estimations of biocontaminants in laboratory cultures; 15. for a rapid check of biopollutants under field conditions; 16. agglutination compositions; 17. a natural colorant; 18. a bioactive composition of the marine dye in the ratio of 1:20000 in ultrapure water to obtain fluorescences of three colors at six different wavelengths and a phase contrast effect under transmitted light; 19. a dye for various fluorescent applications to be performed in areas of sub zero temperatures; 20. for fertilization rate increase in aquaculture industry; 21. for cell permeant membrane dye compositions; 22. for identification of dead and live cells in tissue cultures; 23. for dye compositions in bio-sensor; and 24. as dye composition in molecular and microbiological kits.
- 43. A process for extraction of a natural fluorescent dye from Holothuria scabra sea-cucumber, said process 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) dissecting the animals of step (a) and removing the female gonads, c) extracting the femal gonads of step (c) with 70% ethyl alcohol at least thrice without homogenization of tissues, and d) obtaining the solution containing the fluorescent dye termed as “Stock solution of the dye”.
- 44. The process as claimed in claim 43, wherein the dye is diluted with water in the ratio 1:400,000 times and more and which gives fluorescence of six colors at three different excitation wavelengths.
- 45. The process as claimed in claim 43 wherein the colored part of the dye is attached to a negatively charged protein component.
- 46. The process as claimed in claim 43 wherein the dye is diluted with 70% alcohol to 9000 times and further 1 micro liter is added to per 25-50 micro liter of seawater in drops carrying eggs and bacteria in water in the ratio 1:225,000 times to 1:400000 times and above gives fluorescence of six colors at three different excitation wavelengths
- 47. The process as claimed in claim 43 in which the bioassays were conducted by using dilutions in the range of 1:40000, 1:20000, 1:10000, 1: 5000,and 1:2500 dilutions for assessing nontoxic nature of the dye upon survival of eukaryotic and prokaryotic cells.
Parent Case Info
[0001] The present application is a continuation application of U.S. patent application Ser. No. 09/820,654 filed on Mar. 30, 2001
Continuation in Parts (1)
|
Number |
Date |
Country |
Parent |
09820654 |
Mar 2001 |
US |
Child |
10107335 |
Mar 2002 |
US |