Natural non-polar fluorescent dye from a non-bioluminescent marine invertebrate, compositions containing the said dye and its uses

BACKGROUND AND PRIOR ART REFERENCES

[0003] Sea cucumbers have following taxonomic position.

1Subkingdom: MetazoaPhylum: EchinodermataSub-Phylum: EleutherozoaClass: HolothuroideaSubclasses: Aspidochirotacea, Dendrochirotacea, ApodaceaOrders: Dendrochirota, Aspidochirota, Elasipoda, Molpadoniaand Apoda

[0004] Amongst these orders sea cucumber Holothuria scabra belongs to:

2Order: AspidochirotaFamily: HolothuriidaeGenus: HolothuriaSpecies: scabra

[0005] Echinoderms are coelomate invertebrates which are exclusively marine, never colonial, are unsegmented with a basic pentameric radial, symmetry in the adult form, no head or brain, and distinguished from all other animals by structural peculiarities of skeleton and coelom. Class Holothuroidea has animals with body bilaterally symmetrical, usually elongated in the oral-aboral axis having mriouth at or near one end and anus at or near the other end. The body surface is coarse, endoskeleton reduced to microscopic spicules or plates embedded in the body wall, mouth surrounded by a set of tentacles attached to water vascular system; podia or tube feet are usually present and locomotory; alimentary canal is long and coiled and cloaca usually with respiratory trees; sexes are usually separate and gonad single or paired tuft of tubules. They are sedentary forms either attached to hard substrate or burrow into soft sediments with anterior and posterior ends projected. There are more than 1000 species of holothuroids. They vary from 2 cm to 2 meter in length. They are among a few of the animals whose habitat is not restricted by ocean depths. Some of the species are reported to be making 50% of life forms at 4000 m and 90% at 8000-m depths. The species Holothuria scabra also called by some as Metriatyla scabra Jaegea is widely distributed in East Africa, Red Sea, Bay of Bengal, East India, Australia, Japan, South Pacific, Philippines, Indian Ocean and other Indo-Pacific regions. It is used for human/animal consumption in Sabah, Malaysia and Indonesia and other Indo-Pacific countries.

[0006] Fluorescent dyes are of immense value in the fluorescent probe industries to enable researchers to detect particular components of complex biomolecular assemblies, including live cells with exquisite sensitivity and selectivity (lain D. Johnson. “Introduction to fluorescence techniques” Chapter 1.pp1 in The Handbook of Fluorescent probes and Research Chemicals by Richard P. Haughland, 6th edition Printed in the united States of America, 1996). Fluorescent dyes are widely used in labeling of molecular probes for localizing biological structures by fluorescence microscopy e.g in immunoassays, labeling nucleotides and oligonucleotides for in situ hybridization studies, binding to polymeric microspheres and staining of cells for use in imaging studies. Dyes are also used for selective destruction of cells such as in the technique of photodynamic therapy. (Baughland, R. P and Kang, H. C. U.S. Pat. No. 4,774,339 of Sep. 27,1988; Haughland, R. P and Kang, H. C. U.S. Pat. No. 5,248,782, Sep. 28,1993).

[0007] Non-polar dyes are useful in detection of neutral lipids and other non-polar liquids. The fluorophores with the non-polar quality are most suitable for making conjugants with variety of biomolecules as per the requirement of the reseachers. Two non-polar probes namely NBD probes, NBD dihexadecylamine (D-69) and NBD hexadecylamine (H-429) are primarily been used for fluorescence detection of lipids separated by TLC (The Molecular probes Handbook of Fluorescent probes and Research Chemicals by Richard P. Haughland, 6th edition Printed in the united States of America, 1996 pp.321-322). The same authors reported three non-polar derivatives of the bodipy fluorophore that offers an unusual combination of non-polar structure and long-wavelength absorption and fluorescence. These dyes are described to have potential applications as stains for neutral lipids and as tracers for oil and other non-polar liquids. For example BODIPY 493/503 (D-3922) is more specific for cellular lipid droplets then staining with Nile red (N-1142). BODIPY 505/515 (D-3921) rapidly permeates cell membranes of live Zebrafish embryos, selectively staining cytoplasmic yolk platelets (Mark Cooper, University of Washington quoted by Molecular probes The Handbook of Fluorescent probes and Research Chemicals by Richard P. Haughland, 6th edition Printed in the united States of America, 1996 pp.322). Bodipy dyes have a small fluorescence, Stokes shift extinction coeffecients that are typically greater than 80,000 cm−1 M −1 and high fluorescence quantum yields (0.94 for D-3921 in methanol) that are not diminished in water. Their photostability is generally high These Bodipy dyes are used in flash lamp-pumped laser dyes. Bodipy 665/676 (B-3932) can be excited using the 633 nm spectral line of the he-Ne laser or the 647 nm spectral line of the Ar-Kr laser for applications requiring long-wavelength fluorescence detection. (Richard P. Haughland, Molecular probes. The Handbook of Fluorescent probes and Research Chemicals 6th edition Printed in the United States of America, 1996 pp.321-322). Non-polar fluorescent dyes as dye component in industrial applications are reported in US patents (Fischer, Wolfgang; Deckers, Andreas; Guntherberfg, Norbert; Jahns,Ekkehard; Haremza,Sylke; Ostertag, Werner; Schmidt, Helmut U.S. Pat. No. 5,710,197 released on Jan. 20,1998 entitled “Crosslinked polymer particles containing a fluorescent dye”; U.S. Pat. No. 5,304,493 dated Apr. 19, 1994 by Nowak, Anthony V Title “Method for detecting a marker dye in aged petroleum distillate fuels; U.S. Pat. No. 4,063,878 dated Dec. 20, 1977 by Weeks, Bruce W entitled “Applying sublimation indicia to pressure sensitive adhesive tape”).

[0008] At the website, http://www.uniulm.de/uni/fak/natwis/oc2/ak_wuert/publications.htm there are reported 39 publications of A. K. Wuerthner, most of which pertains to synthesis and design of chromatphores for refractive index materials and photorefractive organic glasses. About nine international patents are also disclosed (S. Beckmann, F. Effenberger, F. Würthner, F. Steybe, DE 44 16 476 (Nov. 16, 1995), WO 95/30679, Preparation of oligothiophenes for use in nonlinear optical materials; K-H. Etzbach, C. Kräh, R. Sens, F. Würthner, DE 19.611.351 (Mar. 22, 1996), WO 97/35926 (Oct. 2, 1997); Farbstoffmischungen, enthaltend Thienyl- und/oder Thiazolazofarbstoffe; Dye mixtures containing thiophene and/or thiazole azo dyes Farbstoffmischungen, enthaltend Thienyl- und/oder Thiazolazofarbstoffe; Dye mixtures containing thiophene and/or thiazole azo dyes; C. Grund, H. Reichelt, A. J. Schmidt, F. Würthner, R. Sens, S. Beckmann, DE 19648564 A1 (May 28, 1998), DE 196 50 958 A1 (Jun. 10, 1998); WO 98/23688 (Jun. 4, 1998) Indoleninmethinfarbstoffe auf Basis von Trifluormethylpyridonen; Trifluormethylpyridone Based Indolenine Methine Dyes; Indoleninmethinfarbstoffie auf Basis von Trifluormethylpyridonen; Trifluormethylpyridone Based Indolenine Methine Dyes; F. Würthner, H.-W. Schmidt, F. Haubner, DE 19643097 A1 (Apr. 23, 1998), Vernetzbare Triarylaminverbindungen und deren Verwendung als Ladungstransportmaterialien; Crosslinkable triarylamines bearing ethynyl groups for use as charge transport materials; F. Würthner, R. Sens, G. Seybold, K.-H. Etzbach, DE 197 11 445 A1 (Sep. 24, 1998), WO 98/41583 (Sep. 24, 1998) Farbstoffsalze und ihre Anwendung beim Färben von polymerem Material; Colorant Salts and their Use in Dying Polymeric Materials )

[0009] At website, www.sigma-aldrich.com and in their section immunochemicals of the catalogue “Biochemicals and reagents for life sciences research, 2000-2001 of Sigma-Aldrich has described labeling reagents and cell linker labeling kits pp. 1454-1456. Three US patents by Horan,Paul K, Jensen, Bruce D, Siezak,Sue E , U.S. Pat. No. 4,783,401 of Nov. 8, 1988; Horan,Paul K, , Siezak,Sue E in U.S. Pat. No. 4,762,701of Aug. 9, 1988 and Horan,Paul K, Jensen, Bruce D, Siezak,Sue E in U.S. Pat. No. 4,859,584 dated Aug. 22, 1989 disclosed respectively “viable cell labeling”, in vivo cellular tracking and cell growth rate determination by measurement of changes in cyanine dye levels in plasma membranes.

[0010] Nontoxic and cell permeant fluorescent dyes are greatly in demand for their applications in the study of live cell functions, drug delivery, study of various cell organelle and many more. An eukaryotic cell may have several compartments each bounded by a membrane whereas a bacterial cell may consist of a single compartment. The permeability specificity is a characteristic of the cell membranes. A good dye is the one which can show the different parts of the cell at one emission and at different emissions (The Handbook of Fluorescent probes and Research Chemicals by Richard P. Haughland, 6th edition Printed in the united States of America, 1996).

[0011] Dyes with fluorophore attached to various proteins have been synthesized and described in Handbook of fluorescent probes and Research chemicals by Richard P. Haughland 1996. Page 126-128. The range from the blue fluorescent Cascade blue and new AMCA -S dyes to the red fluorescent Texas Red dyes and phycobiliproteins is embodied. The company has high lighted Oregon green conjugates, Rhodol Green conjugates; Bodipy conjugates;Eosin labelled secondary reagents; Red fluorescent Rhodamine Rd-X and Texas Red-X conjugates; Phycobiliprotein conjugates; Cascade blue and AMCA-S conjugates.

[0012] All these dyes are synthetic and single one of them emits in one particular spectral range. The combinations of 2-3 dyes are then made for multiple colored experiments. The same company also offers Jasplakinolide, a macro-cyclic peptide isolated from the marine sponge Jaspis jolnstoni as a cell permeant F-actin probe. But it is toxic and exhibits fungicidal, insecticidal, and antiproliferative activity. Most of the currently available dyes in the market are synthetic. Stainfile-Dyes A has given a Dye index of 264 dyes. Out of which 258 are synthetic and only six are natural dyes. (http://members.pgonline.com/˜bryand/dyes/dyes.htm).

[0013] Production of synthetic dyes often require use of strong acids, alkalis and heavy metals as catalysts at high temperatures. This makes the processes and the effluents to be discharged an issue of environment degradation. The dyestuff industry is continuously looking for cheaper and more environmentally friendly routes to existing dyes. (Hobson and Wales, 1998. Green Dyes, Journal of the Society of Dyers and colorists (JSDC), 1998,114,42-44).

[0014] All of the available dyes are not fluorescent. Bitplane products have displayed list of the 123 fluorochromes in the market and their excitation and emission spectrum (http://www.bitplane.ch/public/support/standard/fluorochrome.htm).

[0015] Fluorescence is a phenomenon in which an atom or molecule emits radiation in the course of its transition from a higher to a lower electronic state. It follows Stoke's law, according to which the wavelength of the fluorescent radiation is always longer than that of the excitation radiation. The process of fluorescence is quite different from the phosphorescence and bioluminescence. The term fluorescence is used when the interval between the act of excitation and emission of radiation is very small (10−8 to 10−3 second). In phosphorescence, the time interval between absorption and emission may vary from 10−3 second to several hours (R. Norman Jones, 1966 in: The encyclopaedia of chemistry, 2nd edition, 1966, Pages 435-436). Bioluminescence is the term used for the light produced as a result of a chemical reaction, occurred at a particular time in a particular cell within the body of a living organism.

[0016] A large number of fluorescent dyes are reported in “The Handbook of Fluorescent probes and Research Chemicals” by Richard P. Haughland, 6th edition Printed in the United States of America, 1996. In the same book on pages 1-6, Ian D. Johnson (1996) described in details the process of fluorescence and its methods of detection in certain molecules called iluorophores or fluorescent dyes by him (generally polyaromatic hydrocarbons or heterocycles). The most versatile currently in use fluorescent dyes are Fluorescein, fluorescein based, BODIPY dyes, and their derivatives. The authors have dealt in with the shortcomings of all these dyes and described their preferences of characteristics of dyes. Many derivatives of the fluorescent dyes and their synthesis are disclosed in US patents (Haughland, R. P and Kang, H. C. U.S. Pat. No. 4,774,339 , published on Sep. 27,1988; Haughland, R. P and Kang, H. C. U.S. Pat. No. 5,248,782 of Sep. 28,1993; Kang, H. C. and Haughland, R. P ,U.S. Pat. No. 5,187,288 published on Feb. 16, 1993;; Kang, H. C. and Haughland, R. P in U.S. Pat. No. 5,274,113 of Dec. 28, 1993 and; Kang, H. C. and Haughland, R. P ,U.S. Pat. No. 5,433,896 Jul. 18, 1995 ; Kang, H. C. and Haughland, R. P. U.S. Pat. No. 5,451,663 published on Sep. 19, 1995). Rosenblum Barnett B, Spurgeon S, Lee Linda G, Benson Scott C and Graham Ronald J in international patent No. W00058406, publication date Oct. 5, 2000 reported 4,7-Dichlororhodamine dyes useful as molecular probes.

[0017] R. Norman Jones in The encycopaedia of chemistry, 2nd edition, 1966, Pages 435-436 has described specialty of a good fluorophore. According to him A fluorescent molecule must have a good chromophoric system for absorption of excitation energy and a shielding mechanism to save too rapid dissipation of the excitation energy into vibrational motion before the fluorescence retardation act can occur. He also commented that though the relationship of the molecular structure and the fluorescence of compounds are not well understood there are certain groups, presence of which is associated with fluorescence. For example, in the organic molecule presence of phthalein and aromatic structures such as anthracene and naphthacene are particularly associated with bright fluorescence. Few inorganic compounds fluoresce strongly in the liquid state and in solids, fluorescence is often modified by the presence of trace impurities.

[0018] Pigments belong to the categories of inorganic and organic types. The formers are the inorganic chemistry compounds, which are used for various decorative and painting purposes etc. Organic pigments like organic dyes date back to the ancient times. The use of dyes from plants like Eirazil wood, log-wood, Persian berry indigo and madder are reported from near east and far eastern countries even before Biblical times (George L. Clark, 1966 “Encyclopaedia of chemistry, 2nd ed. Pages 833-835).

[0019] Debra K. Hobsonand David S. Wales describe “Green dyes” which are produced as secondary metabolites from some groups of living organisms like fungi, blue green algae, sea urchins, star fishes, arthropods and coral reef coelenterates (Journal of the Society of Dyers and Colourists (JSDC), 114, 42-44, 1998). These are anthraquinone compounds, historically of crucial importance in the dyestuffs industry.

[0020] Variety of carotenoid pigments are reported from the carotenoid-producing bacterial species (U.S. Pat. No. 5,935,808 published on Aug. 10, 1999, inventors Hirschberg, et al and U.S. Pat. No. 5,858,761, Jan. 12, 1999 inventors Tsubokura, et al). Collin; Peter Donald in his U.S. Pat. No. 6,055,936 published on May 2, 2000 disclosed sea cucumber carotenoid lipid fractions and process. Bandaranayake, W. M. and Des Rocher,A 1999 ( Marine biology 133;163-169) described carotenoid pigments from the body wall, ovaries and viscera of Holothuiria atra from Australia.

[0021] All these colorants and dyes are however not fluorescent. Fluorescent dyes most of which are synthetics are disclosed in several international patents and US patents. These have been used in variety of applications. The amount of patents in this field shows the importance of these dyes. Synthetic parazoanthoxanthin A (m.w.214.2), emitting fluorescence at lambda (em) 420 nm, was found to be a pure competitive inhibitor of cholinesterases. Sepcic K, Turk T, Macek P (Toxicon, 36 (6): 937-940,1998). Welch; David Emanuel (U.S. Pat. No. 5,989,135 released on date Nov. 23, 1999 disclosed a luminescent golf ball.

[0022] White et al. (U.S. Pat. No. 6,110,566, dated Aug. 29, 2000 and WO9920688) described a flexible polyvinyl chloride film that exhibits durable fluorescent colors.

[0023] Dipietro Thomas C (International patent WO9938916) disclosed the use of fluorescent polymeric pigments in variety of paints, inks and textiles. Cramer Randall J (Patent No. EP0206718 published on Dec. 30, 1986) described a composition with fluorescent dye for bleaching and brightening of polymer.

[0024] Leak detection is another utility disclosed by some (Leighley; Kenneth C. U.S. Pat. No. 6,056,162 dated May 2, 2000). Cooper et al. U.S. Pat. No. 6,165,384 published on Dec. 26, 2000 disclose a full spectrum fluorescent dye composition for the same purposes. Lichtwardt et al. U.S. Pat. No. 5,902,749 dated May 11,1999 use fluorescent dye in an automated chemical metering system.

[0025] There are reports on few fluorescent natural dyes available. A green fluorescent protein GFP has been described from the pacific jellyfish, Aequora aequora by Shimomura, O, Johnson, F. H. and Saiga, Y; in Journal of cellular and comparative physiology, 59, 223-239, 1962, Chalfie M in 1: Photochem Photobiol 1995 Oct 62 (4): 651-6 “Green fluorescent protein”; Youvan D C, Michel-Beyerie ME “Structure and fluorescence mechanism of GFP in National Biotechnology Oct. 14, 1996 (10): 1219-20 and Chalfie M, Yuan Tu, Ghia Euskirchen, William W. Ward, Douglas C Prasher in SCIENCE 263 (1994) 802-805 reported that GFP purified is a protein of 238 amino acids. It absorbs blue light maximally at 395 nm with a minor peak at 470 nm and emits green light at the peak emission of 509 nm with a shoulder at 540 nm. This fluorescence is very stable and virtually no photo bleaching is observed.

[0026] GFP with fluorescence in other wavelengths in the ranges of red and yellow are described from non bioluminescent anthozoans, particularly Discosoma coral. Gurskaya N G, Fradkov A F, Terskikh A, Matz M V, Labas Y A, Martynov V I, Yanushevich Y G, Lukyanov K A, Lukyanov S A in 1: FEBS Lett Oct. 19, 2001 ; 507 (1): 16-20n described GFP-like chromoproteins as a source of far-red fluorescent proteins. Wachter R M, Elsliger M A, Kallio K, Hanson G T, Remington S J. In 1: Structure Oct. 15, 1998 ; 6(10): 1267-77 described “Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein”. Fradkov A F, Chen Y, Ding L, Barsova E V, Matz M V,Lukyanov S A “Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence. In 1:FEBS Lett 2000 Aug. 18: 479 (3): 127-30. They describe a novel gene for advanced red-shifted protein with an emission maximum at 593 nm was cloned from Discosoma coral. The protein, named dsFP593, is highly homologous to the recently described GFP-like protein drFP583 with an emission maximum at 583 nm. They developed various mutants of both these genes. A hybrid mutant variant resulted I a mutant variant with a uniquely re-shifted emission maximum at 616 nm. Matz M V, Fradkov A F, Labas Y A, Savitsky A P, Zaraisky A G, Markelov M L, Lukyanov S A. 1: Nat Biotechnolo 1999 Dec: 17(10): 969-73.

[0027] In the south east and south pacific countries sea cucumbers are well known for their use in the health food and drug industry as a food item or ingredient to various drug compositions especially for inflammation of joints, sprains and other therapeutics. Several US and International patents are on record and screened. (Fan Hui-Zeng, Yu Song, Yamanaka E, Numata K, Oka T, Suzuki N, muranaka Y in U.S. Pat. No. 5,519,010 dated May 21, 1996; Weiman, Bernard, U.S. Pat. No. 5,888,514 published on Mar. 30, 1999; Katsukura, Kitazato, Kenji Yamazaki, Yasundo U.S. Pat. No. 5,993,797 dated Nov. 30, 1999; Henderson, R. W; Henderson, T and Hammd, T U.S. Pat. No. 6,255,295 dated Jul. 3, 2001; Shinya U.S. Pat. No. 6,203,827 of Mar. 20, 2001 ; U.S. Pat. No. 5,770,205 patent by Collin Peter Donald published on Jun. 23, 1998 and WO patent no. 0001399 published on Jan. 13, 2000; Kovalev V G, Sementsov V K, Slutskaja , Akulin V N , Timchishina G N in RU 2147239 published on Apr. 10, 2000; Li Zhaoming , Zhu Beiwei CN 1286926 dated Mar. 14, 2001 Qu Jianhong, Song Xiuqin, Zheng Fuqiang CN 1223131 dated Jul. 21, 1999; Wufa Zhuang Wufa, Meizheng Zhuang CN 1142365 dated Feb. 12, 1997; Fang Hua CN1312031 dated Sep. 12, 2001 and Ding Cunyi CN1173290 dated Feb. 10, 1998, Collin Peter Donald W09937314 published on Jul. 29, 1999) . The use of the ingredients originated from sea-cucumber in Anti HIV drug are disclosed (Hoshino Hiroo EP 410002 dated Jan. 30, 1991 and Hoshino Hiroo EP495116 dated Jul. 22, 1992). In view of their importance the animals are tried to be cultivated under captivity (Annie Mercier, S C Battaglene and Jean-Francois Hamel in Journal of experimental Marine Biology and Ecology Volume 249 issue 1: 89-110. 2000 “Settlement preferences and early migration of the tropical sea cucumber Holothuria scabra). Gu Zaishi, Wang Shuhai, Zhou Wei disclosed “Ecological reproducing method for Stichopus japonicus “in patent No. CN1179261 dated Apr. 22, 1998.

[0028] But as all these patents are not directly of relevance to the topic of the present patent so they have been not included as reference.

[0029] Goswami, Usha and Ganguly, Anutosh has filed a patent on a natural fluorescent dye from a marine invertebrate (CSIR, NF-140/2001; U.S. patent application Ser. No.: 09/820654 filed on Mar. 30, 2001). This pertains to the crude extract from Holothuria scabra, which has the fluorescent qualities at three different wavelengths when excited at different UV and visible ranges of the spectra of light. The invention also provides a process of the extraction, purification and characterization of this new dye, which is a partially purified natural dye from a sea cucumber. The utilities of the dye as a epifluorescent stain and non-radioactive fluorescent dye useful for labeling of molecular probes for in situ hybridization studies is described besides several other qualities of the dye as a drug. In this patent prior art, we have dealt in details about the pigments, synthetic dyes and natural dyes from terrestrial plants and microbes. (U.S. Pat. No. 4,452,822 published on Jun. 5,1984, inventors Shrikhande, Anil J., U.S. Pat. No. 5,321,268 of Jun. 14, 1994 by Crosby David A and Ekstrom Philip A; U.S. Pat. No. 5,405,416 published on Apr. 11, 1995 authors Swinton; Robert J, U.S. Pat. No. 5,858,761 published on Jan. 12, 1999, inventors Tsubokura, et al. U.S. Pat. No. 5,902,749 of May 11,1999 inventors Lichtwardt et al . U.S. Pat. No. 5,908,650 published on Jun. 1, 1999 inventors Lenoble, et al. U.S. Pat. No. 5,920,429 published on Jul. 6, 1999 Burns et al. U.S. Pat. No. 5,935,808 on Aug. 10, 1999 of Hirschberg, et al; U.S. Pat. No.5,989,135 of Nov. 23, 1999 inventors Welch; David Emanuel; U.S. Pat. No. 6,055,936 of May 2, 2000 ; Collin; Peter Donald; U.S. Pat. No. 6,056,162 May 2, 2000; Leighley; Kenneth C.; U.S. Pat. Nos. 6,103,006 Aug. 5, 2000 DiPietro; Thomas C.; 6,110,566 Aug. 29, 2000 White et al. 6,140,041 Oct. 31, 2000 LaClair; James J. 6,165,384 Dec. 26, 2000 Cooper et al.; 6,180,154 Jan. 30, 2001 Wrolstad et al. EP0206718 published on Dec. 30, 1986 inventors Cramer Randall J; IE901379 of Jan. 30 1991 Lee Linda G; Mize Patrick D; WO9010044 of Jul. 7, 1990. Swinton; Robert J; AU704112 published on Oct. 7, 1997 inventors Burns David M; Pavelka Lee A; DE19755642 of Jun. 24, 1999 of Weimer Thomas D R.; WO9938919, Sep. 28, 1999 Laclair James J; WO0058406 of Oct. 5, 2000 by Rosenblum Barnett B et al.; WO9938916 of Aug. 15, 2000 inventors DiPietro; Thomas C ; WO9920688 of Aug. 29, 2000 inventors Pavelka Lee et al.; WO9920688 of Aug. 29 2000 inventors

[0030] White et al. The multiple uses oiF fluorescent dyes in the molecular biology research, in industrial applications and in life saving devices etc. are also described. Collin, P. D in his U.S patents of Jun. 23, 1998, Mar. 2, 1999and Nov. 16, 1999 respective U.S. Pat. Nos. as 5,770,205, 5,876,762 and 5,985,330 have described therapeutic properties of various body parts of sea cucumber.

[0031] In another patent application, inventors of the present application i.e. Goswami, Usha and Anutosh Ganguly (U.S. Provisional application No. 60/317,190 filed on Sep. 6, 2001) has described a novel organosilicon Si—O—R type of compound and multiple fluorescent natural dye purified from the body wall extract of a marine invertebrate Holothuria scabira. The compound is a polysacchride fluorochrome having a phenolic fluorophore part and is connected to a silicon matrix around it through the sulphate bonds. This silcon part is an integral part of the core molecule and takes part in the metabolism of the animal. The compound is rich in sulfur. The invention also provides a process for the extraction, purification and characterization of the novel compound and the multiple fluorescent dye from a living marine organism, especially sea cucumber. Further, the compound can be an easily miscible ingredient in compositions of Dye industry, Cosmetic industry and pharmaceutical industries.

[0032] In this patent a large body of literature upon fluorescent dyes reported in the Handbook of Fluorescent probes and Research Chemicals by Richard P. Haughland, 6th edition Printed in the united States of America, 1996 are incorporated. In US and international patents many derivatives of the fluorescent dyes and their synthesis are disclosed (Haughland, R. P and Kang, H. C. U.S. Pat. No. 4,774,339 , published on Sep. 27,1988; Haughland, R. P and Kang, H. C. U.S. Pat. No. 5,248,782 of Sep. 28,1993 ; Kang, H. C. and Haughland, R. P ,U.S. Pat. No. 5,187,288 published on Feb. 16, 1993;; Kang, H. C. and Haughland, R. P in U.S. Pat. No. 5,274,113 of Dec. 28, 1993 and; Kang, H. C. and Haughland, R. P ,U.S. Pat. No. 5,433,896 Jul. 18, 1995 ; Kang, H.C. and Haughland, R. P. U.S. Pat. No. 5,451,663 published on Sep. 19, 1995, Rosenblum Barnett B, Spurgeon S, Lee Linda G, Benson Scott C and Graham Ronald J ,intemational patent No. W00058406, publication date Oct. 5, 2000 reported 4,7-Dichlororhodarnine dyes useful as molecular probes).

[0033] All these references pertain to the present patent also.

[0034] The applicants have adopted a different approach from their earlier patents and those reported by other workers.

[0035] The dyes reported in our earlier patents were though natural but they were toxic. The spectral ranges of emissions at various excitation wavelengths were also different. The dyes were toxic, and not suitable for in situ studies of live cells. Their applications and uses in the biomedical and engineering sciences were different.

[0036] The dye now reported is though also a natural dye but is a non-polar multiple fluorescent flurophore extracted from the female gonads of a non-bioluminescent invertebrate. The marine animal source is a holothurian, sea cucumber called Holothuria scabra which is a new source for a non-polar fluorescent dye. The dye is environmentally friendly, as unlike the synthetic dyes there is no need of intervening steps of strong acids and alkalis in their productions. Unlike the earlier described carotenoid pigments from the sea cucumber ovaries this dye is not a carotenoid pigment. It is a non-polar fluorophcre easily separable from the conjugant biomolecule, which is a negatively charged protein.

[0037] Unlike most synthetic fluorescent dyes, our dye does not need to be mixed with another dye for getting different fluorescence hues at different wavelengths. They themselves emit six different colored fluorescence at three different excitation wavelengths, which can have multiple uses. The cell constituents show a contrasting staining from the background where only dye solution is present.

[0038] The present dye is cell permeani. and permeates through plasma membrane, cytosol, nuclear membrane, nucleoplasm and chromosomes.

[0039] Once the dye attaches to the cell membranes it is stable at the room temperature for months and does not get photobleached and contaminated by microbes. Its fluorescence does not get deteriorated at high and low temperatures unlike extracts of some algae and luminescent organisms.

[0040] The dye is nontoxic to the E.coli bacteria and the sex cells and larvae of estuarine and marine animals. So its effluents will not kill the marine and estuarine animals larval stages. It is a nontoxic and eco-friendly dye.

[0041] Another important feature of the dye is that it shows fluorescence in only the live and fixed tissues. The dead ones are not stained and do not give any fluorescence. One important aspect of the dye is its making compositions and kits for non- radioactive in situ labeling of molecular probes and counter staining. At different wavelength excitations it gives the effect equivalent to color of DAPI, FITC and PI and other marketed fluorescent probes. The dye is a natural multiple fluorescent dye. Actually, this single dye covers the colors of wavelength spectrum of 123 flurochromes presently known in the market (see Bitplane products (Fluorochrome) on the Internet (http://www.bitplane.ch/public/support/standard/Fluorochrome.htm).

[0042] Yet another aspect is its use as a nontoxic fluorochrome stain in epiflourescence microscopy for the live cells. The dye is a natural dye and not synthetic which is permeant through various membranes and stains them differentially. This application provides a simple and quick method of checking cytogenetical preparations for multiple uses like molecular diagnostics using fluorescent in situ hybridization techniques, rapid diagnosis of bio-contamination in tissue cultures, food industry and industrial preparations, flow-cytometry etc.

[0043] Yet another aspect of the dye is its use as a component of the non-radioactive labeling kits for advanced molecular biology applications where protein dyes are needed for studies of live cell functions.

OBJECTS OF THE INVENTION

[0044] The present invention discloses a process of extraction, purification and characterization of a fluorescent dye solution from 70% alcoholic solution of ovarian tissue of a marine echinoderm. This is al natural dye, which has non-polar coloring part. bioactive extract containing a natural non-polar and non-proteinaceous fluorescent dye extracted from alcoholic extract of the ovarian cells and a process of extraction, purification and characterization of a fluorescent pigment from ovarian extract of a marine echinoderm. The pigment is a natural dye, which is a non-polar fluorophore. The dye emits fluorescence in multiple fluorescent excitation ranges of UV and visible light spectra. It further discloses the chemical, physical, epifluorescent microscopic nature of the dye. The nontoxic, cell membrane permeable qualities are seen.

[0045] The main object of the present invention is to provide a natural nontoxic ecofriendly non-polar multiple colored fluorescent dye from the ovarian tissue of sea-cucumber Holothuria scabra.

[0046] Another object of the invention is to provide a process for extraction, partial purification and characterization of the said dye from the marine animal Holothuria scabra which is a non bioluminescent marine invertebrate.

[0047] Another object of the invention is to provide a dye which is cell membranes permeable, giving demarcation of cell partitioning and having a visual effect at single and multiple emission ranges of UV and visible light spectral wavelengths.

[0048] Another object of the invention is to develop a dye with high quantum of fluorescence

[0049] Another object of the invention is to develop a dye with less photobleaching.

[0050] Another object of the invention is to develop a longer time photo stable dye at the room temperature.

[0051] Another object of the invention is to use the dye for checking survival and growth of animal and bacterial cells.

[0052] Yet another object of the invention is to provide compositions employing the dye obtained from the tissues of Holothuria scabra.

[0053] Still another object of the invention is to provide a dye that emits fluorescence in six different wavelength ranges of UV and visible light spectra on particular excitation with three wavelengths.

[0054] Another object of invention is to observe the fluorescence and visible spectroscopic analysis and range of emission wavelengths.

[0055] Yet another object is to observe the six different fluorescence colored emissions of the dye in UV and visible ranges of epifluorescence microscopy cubes.

[0056] Still another object of the invention is to observe the effect of fluorescence staining of the cytogenetical slides to screen chromosomes, cells and tissues by using the dye of the invention.

[0057] Yet another object of invention is to see its nontoxic nature by performing experiments with bacteria.

[0058] Yet another object of invention is to see its nontoxic nature by performing experiments with marine animal cells.

[0059] Yet another object of the invention is its application for checking bacterial contaminations in food industry.

[0060] Yet another object of the invention is to observe the fluorescence and visible spectroscopic analysis and range of emission wavelengths.

[0061] Yet another object of the invention is that the dye is useful as a non-radioactive label of fluorescent molecular probes.

[0062] Still another object of the invention is that the dye does not get quenched fast in the excited light and photobleaching does not occur while screening slides.

[0063] Yet another object of the invention is to observe the fluorescence and visible spectroscopic analysis and range of emission wavelengths.

[0064] Yet, another object of the invention is that the dye is useful as a non-radioactive label of fluorescent molecular probes.

[0065] Another object of the invention is that the dye is highly stable at the room temperature of its own as well as when attached to the cell membranes.

[0066] Still another object of the invention is that its fluorescence quality does not get deteriorated even at extremely high and low temperatures.

[0067] Yet, another object of the invention is to develop kits for non-radioactive labeling of molecular probes and counter staining.

[0068] Yet, another object of the invention is industrial use of the fluorophore for synthesizing biomolecule-conjugants for flow-cytometry, micro-arrays, immunoassays and several other molecular applications as per the requirements of the researcher.

[0069] Still another object is to develop kits containing the fluorescent dye as in situ hybridization non-radioactive labelling kits for molecular probes.

[0070] Still another object is to develop low cost LIVE/DEAD Viability/Cytotoxicity kit for animal cells.

[0071] Still another object is to develop low cost LIVE/DEAD Viability/Cytotoxicity kit for bacteria.

[0072] Still another object is to develop low cost LIVE/DEAD Viability kit for sperms.

[0073] Still another object is to develop low cost LIVE/DEAD Viability kit for yeast cells.

[0074] Still another object is to develop low cost LIVE/DEAD Viability kit for animal cells.

[0075] Still another object is to develop low cost bacterial gram stain and LIVE/DEAD Viability kits.

[0076] Still another object is to develop low cost LIVE bacterial gram stain kit.

[0077] Still another object is to develop low cost bacteria counting kit.

[0078] Still another object is to develop low cost kits for animal cells with simple steps.

[0079] Still another object is to develop kits containing the fluorescent dye for cell culture contamination detection.

[0080] Still another aspect of the invention is the specificity and reliability. The live cells color and give fluorescence and the dead cells do not show any fluorescence.

[0081] Still another aspect of the invention is to use a single dye in the kits in place of presently used 2 dyes for live/dead cells detection.

[0082] Still another aspect of the invention is to develop less expensive and non-hazardous proliferation assays than the radioisotopic techniques.

SUMMARY OF THE INVENTION

[0083] Accordingly, the invention provides a bioactive extract containing a natural non-polar and non-proteinaceous fluorescent dye extracted from alcoholic extract obtained from ovarian cells of a non bioluminescent marine organism called sea-cucumber Holothuria scabra. The invention also provides a process for extraction, isolation and characterization of the said dye, which is a fluorophore detached from a protein conjugant. The said dye is partially purified and is in a solution form. The dye is tested for its nontoxic nature upon animal and bacterial live cells. The dye compositions will be useful as fluorescent epifluorescence microscopic stain, as fluorescent dye ingredient in multiple type of applications in the biomedical, molecular biology, microbiology, cell biology, recombinant technologies etc. and in formulation of new biomolecule conjugarits for applications in fluorescent probes.

DETAILED DESCRIPTION OF THE INVENTION

[0084] After much research, the applicants have now identified a novel natural nontoxic fluorescent dye obtained from tissue of marine animals, especially from invertebrates and more specifically from the sea-cucumber Holothuria scabra.

3Subkingdom: MetazoaPhylum: EchinodermataSub-Phylum: EleutherozoaClass: HolothuroideaSubclass: AspidochirotaceaOrder: AspidochirotaFamily: HolothuridaeGenus: HolothuriaSpecies: scabra

[0085] The invention further provides a natural non-polar multiple fluorescent dyes which is obtained from the ovary of the animal and which is nontoxic to live cells. It also describes the physical and chemical nature of the dye and its stability in direct light, high and low temperatures. The said dye has six colored fluorescent emissions at three different excitation wavelengths of UV and visible light spectrum. The invention also relates to screening of cells under fluorescence microscope for a rapid check of contaminations and cell survival. The invention is also concerned with the uses of the dye as a non-radioactive label of protein, DNA and RNA molecular probes for advanced molecular diagnostics, epi-fluorescence microscopy for single double and multiple staining of chromosomes,, cells and tissues, fluorescence in situ hybridization applications, and biocontamination check, in aquaculture and biomedical sciences for enhancement of fertility, as a component of kits where studies on live cells are required, novel remote sensing devices, underwater probes, life saving devices, mark the location of crashed aircraft, life rafts and Defence equipment for example rockets, various fluorescence applications in sub zero temperature conditions and many more. The dye is environmentally ecofriendly as it does not kill larvae of the estuarine and marine animals.

[0086] The invention describes fluorescent dye obtained from marine animals, which either absorb sunlight for their physiological functions or are exposed to longer duration of sunlight and appear to have evolved mechanisms of fluorescence at different wavelengths. Like the phytoplankton, picoplankton and photosynthetic bacteria absorb sunlight for their photosynthetic functions, the required wavelengths of light spectra are used in the chemical pathways and extra light is emitted following Stoke's law.

[0087] The invertebrate animals who do not have an extra outer armor like a shell and conspicuous defence organs, who have hard and spiny skin, who have a strong endoskeleton formed of ossicles, are sedentary or slow mobility, have long hours of exposures to direct sunlight, live in sand or crevices may show fluorescence.

[0088] The present invention seeks to overcome the drawbacks inherent in the prior art by providing highly efficient and selective methods for extraction, purification and characterization of a dye from a marine invertebrate and its multiple uses in making kits for molecular diagnostics using non-radioactive labels, molecular markers, epiflourescence microscopy, component of new instrumentation devices for land and underwater probes, cosmetic industry, food industries and defense purposes etc.,

[0089] The said marine invertebrate is an echinoderm taxonomically called Holothuria scabra belonging to the class Holothuroidea. The product of the invention is a novel nontoxic multicolor fluorescent protein dye, which is reported for the first time. The animals were collected from the shores of central West Coast of India during low tide, brought to the laboratory and maintained in glass tanks containing seawater of salinity 30-32% per par. The animals were adults and sexually mature. The taxonomic position was identified as above.

[0090] In fact, most of the dyes available are synthetic in nature. There are only 6 types of natural dyes. This includes dyes obtained from all living organisms. The fluorescent dye reported in the present invention is the only one of its specialty of multicolor fluorescence nontoxic, living cell membrane permeant extracted from ovarian tissue of a marine sea cucumber and has abilities to be conjugated to biomolecules.

[0091] As used herein the term dye is used for a dye solution, which does not get decolorized by a reducing agent. The said dye imparts color to the fibre, cellulose, cell membranes etc. It is called a natural dye as the source is from a marine animal found commonly in the nature along shores, shallow and deep waters of the world and is not a synthetic pigment. A fluorescent dye is one, which on excitation at a particular wavelength during the transition from a higher to the lower electronic state within a very short duration it emits light.

[0092] Multiple colored fluorescence means the emission of different colored light when excited at different ranges of wavelengths. It emits blue, yellowish green and orangish red colored hues of fluorescence at excitations with different spectra of UV and visible light.

[0093] Cell membrane permeant means that the dye passes through the pores of the live cell and nuclear membranes of the cell and imparts it multicolored fluorescence in the shades of blue, bluish white, green, yellow orange and orangish red depending upon excitation with the UV, blue and green wavelength ranges.

[0094] The dye does not stain the already dead cells of both animal and bacteria.

[0095] The nontoxic to live cells means when tested upon live cells of both marine animal and bacteria (E.coli), their cells do not die.

[0096] Photostability at room temperature means when left on the bench at room temperature (˜28 degree centigrade) the florescence quality does not deteriorate. Photostabilty of dye after attaching to cell membranes means the continuation of fluorescence emission after staining the live and fixed cells with the said dye.

[0097] The molecular diagnostics as used herein means the use of the dye as a non-radioactive label of molecular probes for fluorescent in situ hybridization applications in molecular cytogenetics and as markers in microarrays, and molecular biological studies.

[0098] The epifluorescent microscopy here pertains to the microscopic studies of cyto-genetical preparations of slides by using the present dye as a stain and recording different colored fluorescence when observed under different cube configurations emits a particular colored emission on excitation with known fluorochromes. The fluoro-chrome cubes WUB, WB, WG are the designated filter cube configurations of the Olympus BX-FLA reflected light fluorescence attachment for different wavelengths.

[0099] Accordingly, the present invention provides a bioactive extract containing non-polar and non-proteinaceous fluorescent dye obtained from alcoholic extract ovarian cells of a non bioluminescent marine organism Holothuria scabra occurring in intertidal, submerged, shallow and deep waters, usually abundant in shaded areas such as acloves, crevices, ledges, overhangings, rocky and sandy habitats; dull to bright coloured with or without exo- and endo skeleton, sessile, sedentary drifters, ektonic with varied swimming power usually nocturnal in habit, liabile to active predation, with and without luminescent and fluorescent pigments giving emissions in few to all wavelength ranges of UVB,UVA visible colored spectrums and infra red spectrum.

[0100] One embodiment of the invention relates, to the bioactive extract obtained from the marine organism, which is useful as a natural fluorescent dye having the following characteristics:

[0101] i. no decolorization by a reducing agent,

[0102] ii. it is a natural compound,

[0103] iii. crude extract of the dye is orange in color,

[0104] iv. semi purified dye solution is bright orange in color for the naked eye,

[0105] v. under tube light it emits variety of colors of the visible light spectrum,

[0106] vi. the pigment separated foim protein part is insoluble in water and alcohol,

[0107] vii. the partially purified dye pigment is soluble in ether.

[0108] viii. the dye is a non-polar dye having pH 7.0,

[0109] ix. absence of reduciable chromophore,

[0110] x. dye solution emits six different colored fluorescence at 3 different wavelengths of the UV and visible ranges of the fluorescent cubes of an epifluorescence microscope, depending upon whether it is the dye solution alone or the cells on which dye has permeated and attached,

[0111] xi. fluorescence of blue color emission occur in the 450 nm -470 nm range on excitation under ultra violet cube WU-330 nm-385 nm excitation range,

[0112] xii. fluorescence of yellowish green color emission occurs in the 510 nm-570 nm range on excitation under WB cube of 450 nm-480 nm excitation range,

[0113] xiii. fluorescence of orange color emission occurs in the 610 nm - 650 nm range on excitation under WG cube of 510 nm-550 nm excitation range,

[0114] xiv. the dye emits hues of yellowish grays under the ordinary transmitted light bulb of the epifluorescence microscope when seen under 100× oil immersion objective,

[0115] xv. the dye emits fluorescence colors even at a dilution range of 1:40000 times

[0116] xvi. the fluorescence of the extract persisted even up to 1 year when preserved at about 4° C.,

[0117] xvii. the fluorescence of the dye is highly photostable and does not get deteriorated by long exposures to direct light,

[0118] xviii. 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,

[0119] xix. the dye is nontoxic to the living cells of eukaryotes and prokaryotes (E. coli)

[0120] xx. the dye is permeable to cell membranes,

[0121] xxi. the dye is impermeable to dead eukaryotic cells and dead prokaryotic cells, and

[0122] xxii. the dye is non degradable even after staining the cell components.

[0123] Another embodiment of the invention, the multicolored emissions of the dye at different wavelengths of excitations are comparable to the fluorochrome microscopic stains available in the market.

[0124] Still another embodiment, 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, immuno-chemistry and molecular biology.

[0125] Still another embodiment, the yellow colored fluorescence of the dye under 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.

[0126] Yet another embodiment, the yellow colored fluorescence of the said dye in the visible range is comparable to the same colored emissions of FHTC used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry and molecular biology.

[0127] Yet another embodiment of the invention provides a dye having orange colored fluorescent emission, comparable to the orange fluorescence color of Propidium Iodide fluorochrome which is ased as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.

[0128] Yet another embodiment of the invention provides a dye having orange colored fluorescent emission, 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.

[0129] Yet another embodiment of the invention provides a dye having orange colored fluorescent emission, 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.

[0130] Yet another embodiment of the invention provides a stable dye at room temperature and has a long shelf life.

[0131] Yet another embodiment of the invention the molecular and radioactive kits of the said dye can be exported at the room temperatures.

[0132] Yet another embodiment of the invention provides a dye having characteristics of at least one hundred different fluorochromes available in the market namely DAPI, Hoechest 33258, Hoechest 33342, FITC, acridine orange, auramine, Rhodamine, TRITC, and propidium iodide, etc.

[0133] Yet another embodiment of the invention provides a dye used in all applications in place of Phycobiliproteins.

[0134] Yet another embodiment of the invention, the said dye 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.

[0135] Yet another embodiment of the invention, the said dye 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, which is useful in rapid bioassays of effect can be seen on the various histochemical components of the cells.

[0136] Yet another embodiment of the invention, the fluorescence color emissions follow Stoke's law of fluorescence.

[0137] Yet another embodiment of the invention, the microphotographs with Kodak film rolls show hues of the adjacent color emission wavelengths such as blue color fluorescence under the epifluorescence.

[0138] Yet another embodiment of the invention, 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 found.

[0139] Yet another embodiment of the invention, orange fluorescence color is seen under the epifluorescence microscope in microphotograph, the hues of red also found.

[0140] Yet another embodiment, 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.

[0141] Yet another embodiment, the diluted dye with water in the ratio above 1:4,50,000 to 1:9,00,000 times give fluorescence of six colors at three different wavelengths.

[0142] One more embodiment of the invention provides compositions comprising bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with suitable additives for the following useful applications:

[0143] i. preparing fluorescent color coating compositions and inks useful in variety of paints, inks, textile;

[0144] ii. as a fluorescent molecular probe in situ hybridization kits for molecular diagnostics;

[0145] iii. used in experiments where various applications of fluorescent dyes are needed to be performed at field stations situated at subzero degree temperature areas;

[0146] iv. useful as fluorochrome stains for epifluorescence microscopy;

[0147] v. useful in cell permeant dye compositions;

[0148] vi. a composition of fluorescent dye for bleaching and brightening polymer;

[0149] vii. leak detection with a full spectrum fluorescent dye;

[0150] viii. use in automated chemical metering system;

[0151] ix. to mark location of crashed air-crafts, life crafts, and equipment for example rockets;

[0152] x. under sea probes;

[0153] xi. chromatophore sunscreen component of cosmetics creams and lotions;

[0154] xii. fluorescent in situ hybridization application kit component for molecular diagnostics;

[0155] xiii. 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;

[0156] xiv. immunofluorescent detections;

[0157] xv. counterstain of DIG-labeled oligonucleotide probes and Anti-DIG Fab-fragments;

[0158] xvi. single and multiple flow cytometry applications;

[0159] xvii. fluorochrome stains for epifluorescence microscopy;

[0160] xviii. for a quick check of bioc,ontamination in the health food industry, cosmetic industry, pharmaceutical and chemical industries;

[0161] xix. for rapid estimations of biocontaminants in laboratory cultures;

[0162] xx. for a rapid check of biopollutants under field conditions;

[0163] xxi. for a rapid check of dead and live bacteria E.coli;

[0164] xxii. a natural colorant;

[0165] xxiii. a bioactive composition of the fluorescent dye in the ratio of 1:400000 in ether is sufficient to produce fluorescences of six colors at three different wavelengths and a phase contrast effect under transmitted light;

[0166] xxiv. a dye for various fluorescent applications to be performed in areas of sub zero temperatures;

[0167] xxv. a dye for making conjugants with variety of biomolecules;

[0168] xxvi. for cell permeant membrane dye compositions;

[0169] xxvii. for identification of dead and live cells in tissue cultures;

[0170] xxviii. for dye compositions in biosensors;

[0171] xxix. as dye composition in molecular and microbiological kits; and

[0172] xxx. a non-polar dye for detection of lipids and oils.

[0173] One more embodiment of the invention relates to a process for preparation of a bioactive extract containing a natural fluorescent dye from Holothuria scabra sea cucumber, said process comprises the steps of:

[0174] a) collecting the marine organism from seashore,

[0175] b) maintaining the organism in tanks containing seawater without any mechanical aeration overnight,

[0176] c) dissecting the washed animals and removing the female gonads,

[0177] d) extracting with 70% ethyl alcohol to obtain yellowish orange solution,

[0178] e) repeating the extraction steps 3-4 times to obtain colored solution,

[0179] f) filtering the above solution through Whatman No.1 filter paper to obtain partially purified extract,

[0180] g) dissolving the extract obtained in step (f) in solvent ether, and

[0181] h) filtering through Whatmaini No 1 filter paper to obtain clear yellow colored solution named as “Non-polar fluorescent dye solution

[0182] Another embodiment of the invention, the said dye solution is diluted further with water/seawater, in the ratio ranging between 1:400,000 to 1:900,000 times give fluorescence of six colors at three different excitation wavelengths.

[0183] Still another embodiment of the invention, wherein bioassays are conducted by using dilutions of the bioactive extract in the range of 1:450000, 1:200000, 1:100000, 1:50000 and 1:25000 times for assessing non-toxic nature of the dye upon survival of eukaryotic and prokaryotic cells.

[0184] The present invention provides a method for extraction, partial purification and characterization of a natural non-polar nontoxic cell membrane permeant multiple fluorescent dye. It further provides compositions at which the dye can detect live eukaryotic and prokaryotic cells without killing them and it comprises of:

[0185] collection of the material from field and maintenance in the laboratory conditions,

[0186] extraction of the pigment from the 70% alcoholic extract of the ovary of the echinoderm sea cucumber

[0187]

Holothuria scabra
, and partial purification of the dye.

[0188] Testing of biological activities.

[0189] The bioactive extract of the invention is obtained from 70% alcoholic extract of the ovarian tissue of marine sea-cucumber Holothuria scabra. This extract is useful as a natural fluorescent dye and has the following characteristics:

[0190] 1) no decolorization by a reducing agent,

[0191] 2) not a synthetic compound,

[0192] 3) crude extract of the dye is yellowish orange in color,

[0193] 4) the dye has two steps of its extraction from the ovarian cells i.e. at step-i the alcoholic solution is extracted and left to evaporate and step-2, solvent ether is added to the dried mass and filtered through Whatmann filter 1.

[0194] 5) it produces dye solution containing partially purified fluorophore.

[0195] 6) the dye solution is bright orange in color.

[0196] 7) under tube light it emits variety of colors of the visible light spectrum.

[0197] 8) the pigment is insoluble in water and alcohol.

[0198] 9) the pure dye pigment is soluble in solvent ether, the further dilutions can be made by following the procedures given.

[0199] 10) is a non-polar dye,

[0200] 11) has pH of about 7,

[0201] 12) absence of a reducable group,

[0202] 13) the fluorophore is associated with the protein which is removed when heated and coagulated,

[0203] 14) dye in solution emits six different colored fluorescence at 3 different wavelengths of the UV and visible ranges of the fluorescent cubes of an epifluorescence microscope, depending upon whether it is the dye solution alone or the cells on which it has attached.

[0204] 15) fluorescence blue color emission occur in the 450 nm -470 nm range when excited under ultra violet cube NVU - 330 nm -385 nm excitation range,

[0205] 16) fluorescence yellowish green color emission occurs in the 510 nm-570 nm range when excited under WB cube of 450 nm-480 nm excitation range,

[0206] 17) fluorescence orange color emission occurs in the 610 nm-650 nm range when excited under WG cube of 510 nm-550 nm excitation range,

[0207] 18) the dye emits hues of yellowish grays under the ordinary transmitted light bulb of the epifluorescence microscope when seen under 100× oil immersion objective,

[0208] 19) the dye emitted these fluorescence colors even at a dilution range of 1:400000 to 1:900,000 times and above.

[0209] 20) the fluorescence of the extract persisted even after at least 1 year at the 4 degree centigrade

[0210] 21) the fluorescence of the dye is highly photostable and does not get deteriorated by long exposures to direct light once the cells are stained even at the room temperature.

[0211] 22) the fluorescence of the dye does not change even when frozen at minus 20 degree centigrade, a temperature at which the molecules are unable to attain the energy necessary for activation like in extracts from luminescent organisms.

[0212] 23) The dye is nontoxic to the living cells of eukaryotes.

[0213] 24) The dye is also nontoxic to the prokaryotes (E.coli)

[0214] 25) The dye is cell membrane penmeant

[0215] 26) The dye is cell membrane impermeable to dead animal cells.

[0216] 27) The dye is cell membrane impermeable to dead E.colibacteria.

[0217] 28) The dye is a non-degradable stain of the cell membranes.

[0218] The physical and other characteristics of the dye may be assessed by the following step:

[0219] a. color and solubility of the dye,

[0220] b. photostability

[0221] c. phtobleaching

[0222] d. nontoxicity

[0223] e. selective staining of cell membranes

[0224] f. Physical checking of emission under a UV transilluminator 260-280 nm range,

[0225] g. Preparation of slide with live oyster eggs and sperms

[0226] h. Preparation of slides of live bacteria

[0227] i. Preparation of the fixed cells slides by air dried method,

[0228] j. Staining of slides with the dye,

[0229] k. Keeping controls for each experiment without adding dye.

[0230] l. Epifluorescent microscopic screening of the live eukaryotic cells slides under fluorochrome cubes WU, WB, WG and Bright field,

[0231] m. Epifluorescent microscopic screening of the fixed eukaryotic cells under fluorochrome cubes WU, WB , WG and Bright field,

[0232] n. Epifluorescent microscopic screening of the dead eukaryotic cells under fluorochrome cubes WU, WB , WG and Bright field,

[0233] o. Epifluorescent microscopic screening of the live bacterial cells under fluorochrome cubes WU, WB , WG and Bright field,

[0234] p. Epifluorescent microscopic screening of the dead bacterial cells under fluorochrome cubes WU, WB , WG and Bright field,

[0235] q. Epifluorescent microscopic screening of the live control cells without the dye under fluorochrome cubes WU, WB, WG and Bright field,

[0236] r. Microphotography of emitted fluorescence in the areas of slides without any cells,

[0237] s. Microphotography of emitted fluorescence of the cytogenetic slides under fluorochrome cubes WU, WB , WG and Bright field, and

[0238] t. Checking of wavelength ranges of the fluorescent hues of emission and wavelength ranges of the excitation ranges of fluorochrome cubes with the dye

[0239] u. Checking of wavelength ranges of the fluorescent hues of emission and wavelength ranges of the excitation ranges of fluorochrome cubes with the cells stained with the dye

[0240] v. Checking for cell membrane permeability of plasma membrane, cytoplasm, nuclear membrane, nucleoplasm and chromosomes.

[0241] Thus the invention provides a non-polar natural fluorescent dye of marine animal origin which emits six different colored fluorescence in the hues of blue, yellow and orangish red when excited with three different ranges of wavelengths in the UV and visible light spectral cubes of an epifluorescence microscope . The range of emissions of the dye solution and that of cells stained with the dye differs. The invention further relates to the epifluorescence microscopy of eukaryotic and prokaryotic live, fixed and dead cell preparations by using this dye as the epifluorescent microscopic stain. This dye could be used in making non-radioactive labeling kits for molecular diagnostics by fluorescent in situ hybridization in various molecular, biomedical and engineering sciences.

[0242] In an embodiment the source of the dye is an invertebrate marine animal belonging to SubKingdom: Metazoa, Phylum Echinodermata; subphylum: Eleutherozoa, Class Holothuroidea. Name: Holothuria scabra

[0243] In yet another embodiment the Holothuria scabra is selected from the group comprising of sea cucumbers and widely distributed in the shores, shallow waters, deep waters all over the world particularly Indo-Pacific. The nearest well-known relatives of sea cucumber are the sea urchins and starfishes etc.

[0244] In yet another embodiment Holothuria scabra is dissected, its ovaries are separated and weighed. In step 1, to 1 mg of ovarian tissue by wet weight 3 ml×3 times of 70% alcohol is added. This is called 70% alcoholic extract of ovarian tissue.

[0245] In yet another embodiment in step 2, heating the solution coagulates the protein and alcohol is evaporated. The dried mass dissolves only in non-polar solvent ether.

[0246] In yet another embodiment, the colored solution is partially purified by filtering through Whattman No. 1 filter paper.

[0247] In yet another embodiment the vial with screw cap carrying the solution of the dye is labeled as “non-polar dye solution” and stored in cold room at 40 centigrade.

[0248] In yet another embodiment, the color of the dye solution is noted with naked eyes and tube light.

[0249] In yet another embodiment, the dye is found to be soluble in ether. The further dilutions for experiments can be made following given procedures.

[0250] In yet another embodiment, the photostability of the dye at the room temperature in the solution and on the stained cells is noted. It showed that the dye is non-degradable both at the room temperature and also once it attaches to the cell membranes.

[0251] In yet another embodiment test for electric charge of the Dye by electrophoresis is performed, the yellow spot showed non-polar behaviour and does not move towards any pole.

[0252] In yet another embodiment, the pigment is a dye as it is giving a color to the filter paper and staining the cell membranes.

[0253] In yet another embodiment, the dye has a pH of 7.0.

[0254] In yet another embodiment 1 ml of the dye solution of 70% alcoholic extract from which the present dye is isolated was taken and 0.2 gms of di thio erythritol was incorporated. The solution does not get decolorized. It showed that the reducible group is absent in the coloring part of the solution.

[0255] In yet another embodiment 9 ml of the 70% alcoholic extract from which the present dye is isolated was subjected to heating in a water bath at 100-degree Centigrade. Coagulation was observed. Which confirmed the presence of protein in the step 1 solution of the dye.

[0256] In yet another embodiment, after removal of the protein from the starting material, the colored part gets separated and dissolves only in ether.

[0257] In yet another embodiment, the alcoholic extract was subjected to Anthrone test (Ref) by using 4 ml of sulfuric acid and Anthrone reagent. 1 ml of Water 4 ml of conc. sulfuric acid and Anthrone reagent was kept as a blank. The blank was faintly green after 5 minutes whereas the extract was turned bright green after 5 minutes. The test proved that the carbohydrate is present in the alcoholic extract.

[0258] The fluorescence activity of the dye solution is due to a colored component that includes non-polar fluorophore attached to a negatively charged protein, which gets easily separated when the protein is coagulated.

[0259] In yet another embodiment nontoxicity test of the dye on eukaryotic cell survival was performed.

[0260] The dye was tested for cytotoxicity upon the oyster sperms. The survival of the sperms in the experimental set ups was taken as a parameter for showing nontoxicity. Male gonads of an oyster were removed and sperms were released in 100 % seawater. These were filtered through muscline cloth to remove any debris. 1 mililiter (ml) of the sperm solution was Oaken and different concentrations (1 μl, 2 μl, 3 μl, 4μl and 5 μl) of the Dye solutions were added. At every half an hour the observations of survival of the sperms were made under a microscope. The experiments were continued for 24 hours. The Controls were maintained without addition of Dye. It was seen that there was no effect upon the survival rates of sperms with addition of the dye. This proved nontoxic nature of the dye.

[0261] In yet another embodiment nontoxicity test of the dye was performed upon survival of prokaryotes. The extract was tested for cytotoxicity upon gram negative E.coli bacteria by observing their survival or mortality. A drop of the live E.coli bacteria in water (50 μl) was placed on a microscopic slide. To this was added 0.5 μl-1.0 μl of the ether extract. The mixture was mixed using stirring needle. The slide was sealed so as to save it from evaporation. It was seen under the microscope that the bacteria remained alive for 24 hours till they remained in solution. They died if the solution got dried. The control experiments were performed. This proved that the dye is nontoxic to the bacteria.

[0262] In yet another embodiment, the bacteria do not show agglutination behavior. In another embodiment, the oyster eggs, sperms do not show agglutination behavior.

[0263] The applicants studied the nature of the dye and found that it gave multicolored emissions at different wavelengths of excitations, which are comparable to the fluorochrome microscopic stains already in the market. 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. The yellow colored fluorescence of the said dye in the visible range is comparable to the same colored emissions of auramine used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.

[0264] 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.

[0265] 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.

[0266] The orange colored fluorescent. emission is comparable to the orange fluorescence color of TRITC fluorochrorrie used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.

[0267] The dye is stable at the room temperature and has a long shelf life. The molecular and radioactive kits of the said dye can be exported at the room temperatures. The dye has characteristics of at least one hundred and twenty three different fluorochromes namely DAPI, Hoechest 33258, Hoechest 33342, FITC, acridine orange, auramine, Rhodamine, TRITC, and propidium iodide etc., which are now in the market ( Bitplane products). The dye, under ordinary light of microscope the hues of 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. The fluorescence color emissions follow Stoke's law of fluorescence.

[0268] In yet another embodiment the epifluorescence microscopic studies are made by using this dye as a stain in the dilutions of above 1:400000 and recording emissions of light when excited by different cubes and compared the color hues with the known fluorochromes.

[0269] In yet another embodiment the screening was done using excitations of UV light and visible light spectra by WU, WB, WG and BF cubes of the Olympus reflected light.

[0270] In yet another embodiment WU cube's wavelength range is 330 nm-385 nm.

[0271] In yet another embodiment WB cube's wavelength range is 450 nm-480 nm .

[0272] In yet another embodiment WG cube's wavelength range is 510 nm-550 nm.

[0273] In yet another embodiment BF is for the bright field where an ordinary tungsten bulb delivers light.

[0274] In yet another embodiment, the emission ranges of the dye at different excitation ranges were found out. The background of the eggs in the epifluorescence microscopy photos shows the emission color of the dye.

[0275] In yet another embodiment it was seen that excitation with the WU 330 nm-385 nm range emitted fluorescence in the 450 nm-470 nm range.

[0276] In yet another embodiment Excitation with the WB filter having spectral range of 450 nm-480 nm emitted fluorescence in the 510-nm-570 nm range.

[0277] In yet another embodiment, the excitation with the WG filter having spectral range of 510 nm-550 nm emitted fluorescence in the 610-nm-650 nm range.

[0278] In yet another embodiment With BF the shades of yellowish grays were seen.

[0279] In yet another embodiment, the emission ranges of the dye after staining to the cell membranes at different excitation ranges were found out.

[0280] In yet another embodiment, the dye was used as fluorescence microscopic stain on the dead, live and fixed eggs of the oyster. The slides were screened under a epifluorescence microscope. It was noticed that the dead cells donot take up dye and shows no fluorescence.

[0281] In yet another embodiment, the dye was used as fluorescence microscopic stain on the live eggs of the oyster. The slides were screened under a epifluorescence microscope. It was noticed that the live cells showed fluorescence. The excitation spectral range and the emitted fluorescence strictly followed the Stoke's law. These ranges were different from the emission ranges of the dye, which represents the background of the fluorescing cell.

[0282] In yet another embodiment the dye was used as fluorescence microscopic stain on the fixed eggs of the oyster in 3:1 ethanol and acetic acid fixative. The slides were screened under a epifluorescence microscope. It was noticed that the fixed cells showed fluorescence. The excitation spectral range and the emitted fluorescence strictly followed the Stoke's law. These ranges were different from the emission ranges of the dye, which represents the background of the fluorescing cell.

[0283] In yet another embodiment excitation with the WU 330 nm-385 nm range emitted fluorescence in the 470nm-500 nm range in the cells.

[0284] In yet another embodiment excitation with the WB filter having spectral range of 450 nm-480 nm emitted fluorescence in the 570 nm-610 nm range.

[0285] In yet another embodiment, the excitation with the WG filter having spectral range of 510 nm-550 nm emitted fluorescence in the 610 nm-650 nm range.

[0286] In yet another embodiment the epifluorescent microscopic screening of the dead eggs under Bright Field emitted light in full white range of the visible spectra and depending upon the density of the cell ingredients gave hues of yellowish grays like a phase contrast effect.

[0287] In yet another embodiment the epifluorescent microscopic screening of the live eggs under Bright Field emitted light in full white range of the visible spectra and depending upon the density of the cell ingredients gave hues of yellowish grays like a phase contrast effect.

[0288] In yet another embodiment the epifluorescent microscopic screening of the fixed eggs ( 3:1 ethanol:acetic acid fixative) under Bright Field emitted light in full white range of the visible spectra and depending upon the density of the cell ingredients gave hues of yellowish grays like a phase contrast effect.

[0289] In yet another embodiment, the dye was used as microscopical stain for the E.coli. One loop of live E.coli bacteria was placed in 50 microliter of water on a microscopic slide and mixed. To this was added 0.5 -1 microlitre (1 μl) of the dye solution towards outskirt of the bacterial suspension drop. The ether in the extract was allowed to evaporate by leaving the slide on bench for 4-5 sec. Both the drops were then mixed and a coverslip was placed on the sample and immediately sealed. Similarly a control preparation of bacterial suspension in water without dye was made.

[0290] In yet another embodiment both the slides were screened under oil immersion objective of a epifluorescence microscope (100× objective, 10× eye lens) for checking fluorescence. It was noticed that the dead cells don't take up dye and shows no fluorescence.

[0291] In yet another embodiment the live bacterial cells showed fluorescence. The excitation spectral range and the emitted fluorescence wavelengths strictly followed the Stoke's law. These were different from the dye solution and was as given below:

[0292] In yet another embodiment excitation with the WU 330nm-385 nm range emitted fluorescence in the 470nm-500 nm ranges.

[0293] In yet another embodiment Excitation with the WB filter having spectral range of 450 nm-480 nm emitted fluorescence in the 570nm-610 nm range.

[0294] In yet another embodiment, the excitation with the WG filter having spectral range of 510 nm-550 nm emitted fluorescence in the 610nm-650 nm range.

[0295] In yet another embodiment, the control E.coli without any dye also showed no fluorescence.

[0296] In yet another embodiment microphotography of the slides with the dye used as epifluorescence microscopy stain was done.

[0297] In yet another embodiment The microphotography of emitted fluorescence in the areas of slides with cells and the surrounding background which represented the emissions of colors only due to dye was done, under WU 330 nm-385 nm range, with Kodak film of 400 ASA speed with an exposure time varying from 50 to 60 seconds.

[0298] In yet another embodiment the microphotography of emitted fluorescence in the areas of slides with cells and the surrounding background which represented the emissions of colors only due to dye was done, under WB 450 nm-480 nm range, range, with Kodak film of 400 ASA speed with an exposure time varying from 50 to 60 seconds.

[0299] In yet another embodiment the microphotography of emitted fluorescence in the areas of slides with cells and the surrounding background which represented the emissions of colors only due to dye was done, under WB 510 nm-550 nm range, with Kodak film of 400 ASA speed with an exposure time varying from 50 to 60 seconds.

[0300] In yet another embodiment the microphotography of emitted fluorescence in the areas of slides with cells and the surrounding background which represented the emissions of colors only due to dye was done, under Bright field with Kodak film of 400 ASA speed with an exposure time varying from 50 to 60 seconds.

[0301] In yet another embodiment permeation of dye in the Cell membranes was found out. The unfertilized, fertilized eggs and larvae of oysters were stained with the dye and egg suspension ratio of 1:50 microliter and screened under a fluorescent microscope. It was seen that the fluorescence was noticeable in the plasma membrane, nuclear envelope and chromatin. Though the wavelength ranges of emissions were the same and the colors were the hues of the same shade, there was a noticeable demarcation of boundaries of these parts of the cell. This proved that the dye is permeable through the live and fixed cell membranes of egg plasma membrane, cytoplasm, nuclear membrane, nucleoplasm and chromatin.

[0302] In yet another embodiment, the absence of fluorescence of these parts of the cell in the dead cells showed that dye is irnpermeant to dead cell membranes.

[0303] Different stains are used for different excitation cubes of the fluorescent microscope. For example DAPI (DNA staining, emits blue color), Fluorescein-dUTP; Hoechest 33258, 33342 are seen under excitation with 330 nm-385 nm excitation cubes; FITC, Acridine Orange (for DNA, RNA emits greenish/yellowish hues), Auramine under 450 nm- 480 nm excitation cube and Rhodamine, TRITC and Propidium iodide (DNA, emits orange hues) under 510 nm-550 nm excitation cube.

[0304] In an embodiment to this epifluorescence putting a drop of the diluted extract and excitation with the WU filter having spectral range of 330-385 nm wavelengths does microscopic screening of the cytological slides.

[0305] In another embodiment epifluorescence microscopic screening of the cytological slides is done by putting a drop of the extract and excitation with the WB filter having spectral range of 450 nm-480 nm wavelengths.

[0306] In another embodiment epifluorescence microscopic screening of the cytological slides is done by putting a drop of the extract and excitation with the WG filter having spectral range of 510 nm-550 nm wavelengths.

[0307] In yet another embodiment epifluorescent microscopic screening of the cytological slides under Bright Field objective using this dye by transmitted light.

[0308] In yet another embodiment epifluorescence microscopic screening of the cytological slides stained with the dye is done by observing hues of the fluorescence color emitted by the respective excitations.

[0309] In another embodiment the excitation with the WU 330 nm-385 nm range emitted fluorescence is in the 470 nm-500 nm range.

[0310] In another embodiment, the excitation with the WB filter having spectral range of 450 nm-480 nm emitted fluorescence in the 570 nm-610 nm range.

[0311] In yet another embodiment the excitation with the WG filter having spectral range of 510 nm-550 nm emitted fluorescence in the 610 nm-650 nm range.

[0312] In another embodiment epifluorescent microscopic screening of the cytological slides under Bright Field by using transmitted light emitted light in full white range of the visible spectra depending upon the density of the cell ingredients and giving a phase contrast effect.

[0313] In still another embodiment, the dye is diluted in 70% ethyl alcohol 1:9000 times and vaporized and the dried matter is again dissolved in 3 ml ether by repeating dissolution in the same amount of ether three times and further mixed in water to 1:50 time which means total dilution of the pigment is above 1:400000 times which gives fluorescence of six colors at three different wavelengths.

[0314] In yet another embodiment the invention provides a bioactive composition containing an extract obtained from the marine sea-cucumber Holothuria scabra in the ratio of 1:400000 to obtain fluorescence of six colors at three different wavelengths and a phase contrast effect under transmitted light.

[0315] In an embodiment, the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful in the preparation of coating compositions and inks.

[0316] In another embodiment, the invention provides composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful in detection of leaks.

[0317] In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful in undersea probes.

[0318] In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful as a fluorescent probe in situ hybridization kits for molecular diagnosis.

[0319] In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria 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.

[0320] In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful in immuno fluorescent detections.

[0321] In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful as a counterstain of DIG-labeled oliogonucleotide probes and anti-DIG Fab-fragments.

[0322] In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful in single and multiple cell quantitative fluorescence in flowcytometry.

[0323] In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful as fluorochrome stains for epifluorescence microscopy.

[0324] In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful for a quick check of biocontamination in the health food industry, cosmetic industry, pharmaceutical and chemical industries.

[0325] In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful for rapid estimations of biocontaminants in laboratory cultures.

[0326] In yet another embodiment the invention provides a composition comprising a bio active extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful for a rapid check of biopollutants under field conditions.

[0327] In yet another embodiment the invention provides a composition comprising a bio active extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful in microbial kits.

[0328] In yet another embodiment the invention provides a composition comprising a bio active extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful as a natural colorant.

[0329] In yet another embodiment the dye solution contains a fluorophore which can be conjugated with proteins and other bio molecules for making customer oriented dye compositions.

DESCRIPTION OF THE TABLES

[0330] TABLE-1 The Emissions of the different colored fluorescence of the fluorescent dye when excited with different wavelength fluorescent filter cubes of the Olympus epifluorescence microscope with dye solution and when attached to cell membranes of the prokaryotic cells.

[0331] TABLE-2 The Emissions of the different colored fluorescence of the fluorescent dye when excited with different wavelength fluorescent filter cubes of the Olympus epifluorescence microscope with dye solution and when attached to cell membranes of the eukaryotic cells.

[0332] Table-3 Table showing various commercially important features of the three natural fluorescent dyes extracted from the sea cucumber of the present invention.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:

[0333]
FIG. 1 Black and white figure of epifluorescence microscopy results of green color fluorescent emissions of the dye and yellow colored emissions of the bacterial cells where dye has attached (arrow) when the dye was excited with WB filter cube of Olympus BX-60 microscope having excitation range of 450-480 nm.

[0334]

FIG. 1

a
Colored photograph of FIG. 1.

[0335]
FIG. 2 Black and white figure of epifluorescence microscopy results of dark red color fluorescent emissions of the dye and orange colored emissions of the bacterial cells where dye has attached (arrow) when the dye was excited with WG filter cube of Olympus BX-60 microscope having excitation range of 510 nm-550 nm nm.

[0336]

FIG. 2

a
Colored photograph of FIG. 2.

[0337]
FIG. 3 Black and white figure of epifluorescence microscopy results of dead bacteria showing no fluorescence.

[0338]

FIG. 3

a
Colored photograph of the same under WG filter cube of Olympus BX-60 microscope having excitation range of 510 nm-550 nm.

[0339]
FIG. 4 Black and white figure of epifluorescence microscopy results of dark red color fluorescent emissions of the dye and orange colored emissions of the oyster egg cell where dye has attached when the dye was excited with WG filter cube of Olympus BX-60 microscope having excitation range of 510 nm-550 nm nm. The nuclear envelope and chromatin is seen.

[0340]

FIG. 4

a
Colored photograph of FIG. 4.

[0341]
FIG. 5 Black and white figure of epifluorescence microscopy results of green color fluorescent emissions of the dye and yellow colored emissions of the live egg cells where dye has permeated (arrow) when the dye was excited with WB filter cube of Olympus BX-60 microscope having excitation range of 450-480 nm. The cell permeant nature of the dye showing egg membrane and nuclear envelope and the cytoplasm and nucleoplasm stained (arrows)

[0342]

FIG. 5

a
Colored photograph of FIG. 5. The chromatin of sperm nucleus is also seen.

[0343]
FIG. 6 Black and white figure of epifluorescence microscopy results of dark bluish green color fluorescent emissions of the dye and blue colored emissions of the live egg cells where dye has permeated (arrow) when the dye was excited with WU filter cube of Olympus BX-60 microscope having excitation range of 330 nm-385 nm. The cell permeant nature of the dye showing egg membrane and nuclear envelope and the cytoplasms and nucleoplasm stained.

[0344]

FIG. 6

a
Colored photograph of FIG. 6. The chromatin inside the nucleus is also seen.

[0345]
FIG. 7 Black and white figure of epifluorescence microscopy results of dark bluish green color fluorescent emissions of the dye and bright bluish white colored emissions of the oyster embryo cells 5 days after the permeation of the dye (arrow) when the dye was excited with WU filter cube of Olympus BX-60 microscope having excitation range of 330 nm-385 nm.

[0346]

FIG. 7

a
Colored photograph of FIG. 7.

[0347]
FIG. 8 Black and white figure of epifluorescence microscopy results of dark bluish green color fluorescent emissions of the dye and yellow colored emissions of the live oyster embryo cells 5 days after permeation of the dye (arrow) when the dye was excited with WB filter cube of Olympus BX-60 microscope having excitation range of 450-480 nm.

[0348]

FIG. 8

a
Colored photograph of FIG. 8.

[0349]
FIG. 9 Flow-chart.

[0350] This invention pertains to the process of extraction, partial purification and characterization of a New natural non-polar pigment which is fluorescent dye from an echinoderm (Holothuroidea: Holothuria scabra) widely distributed along the central West Coast of India and the Indo-pacific regions of the world.

[0351] The invention further provides Ma novel fluorescent protein dye from the ovarian tissue of the animal which can be repeatedly extracted 3-4 times from the same tissue by storing in 70% ethyl alcohol under −4 degree Centigrade, thus saving over exploitation of natural resources.

[0352] The present invention also contemplates that the said dye has six colored fluorescent emissions at three different excitation wavelengths of UV and visible light spectra equivalent to emissions by six different fluorochromes (DAPI, FITC and PI) and the pycobiliproteins and rhodamines currently used for multicolor fluorescent detentions. The dye actually covers the wavelength emission spectra of about one hundred and twenty three fluorochromes currently sold in the market for fluorescent microscopy probes. The stained cells show different color emissions at the same excitations but strictly follow stroke's law. Thus altogether at same excitations of olympus BX-60 microscope filters the dye emits 6 colors.

[0353] The dye is nontoxic to living E.coli bacteria and eukaryotic cells.

[0354] The dye permeates through the various cell constituents membranes and stains them fluroscently.

[0355] The dye is non-degradable once it is attached to cell membranes by staining them. The dye does not show agglutination of bacteria and anomal cells. The dye is a fluorophore in ether solution. The fluorciphore is conjugated to a protein in the 70% alcohol solution of ovarian tissue.

[0356] Thus, the dye can be commercialized as natural nontoxic cell permeant multiple fluorescent dye of epifluorescence microscopy for single, double and triple staining of chromosomes cells and tissues following simple protocols.

[0357] The present invention also contemplates the use of the dye in non-radioactive labeling of protein, DNA and RNA probes for fluorescent in situ hybridization applications in molecular biology.

[0358] Thus in a preferred mode of use the dye can be a component of molecular labeling and detection kits, most of which are imported and sold at high rates.

[0359] These labeling kits are widely sought after for molecular diagnostics using rapid molecular cytogenetic and microarrays techniques.

[0360] Yet another advantage of the dye is that its fluorescence is visible even in very dilute solutions (1:40000 to 400000) and above.

[0361] This property and the nontoxic Eco friendly nature of the dye can be utilized in life saving devices as a component of life jackets and to mark the location of crashed aircraft, life rafts and defense equipment for example rockets, leaking checks in the industries etc.

[0362] The invention would be useful for quantitative measure of fluorescence in flow cytometer for single and multiple cells.

[0363] The invention would be also advantageous in quick estimations of bio-contamination in natural and controlled environments like tissue cultures, pollution, and industrial contamination in health, food and cosmetic industries.

[0364] In another preferred mode of use the dye bottle if sealed properly has a long shelf life at the room temperature.

[0365] In another preferred mode of use the dye has a long shelf life at the room temperature once the cells are stained as checked by fluorescent microscopic analysis.

[0366] Another utility of the fluorescent dye is as a component of novel remote sensing devices and undersea probes where light wavelength sensitivity based data is required.

[0367] The invention is illustrated by the following examples, which should not be construed as limitations on the inventive scope of the invention in any manner.

EXAMPLES

[0368] The methods of isolation, partial purification, characterization of the dye and the details of the experiments performed to check non-polar, nontoxic, cell permeant, non-agglutination and multiple fluorescent effects of the dye by epifluorscence microscopy are disclosed:

Example 1

[0369] Collection of the material:

[0370] Material of the patent is a sea cucumber with following taxonomic details.

[0371] Subkingdom: Metazoa ; Phylum: Echinodermata; Sub-Phylum: Eleutherozoa; Class:

[0372] Holothuroidea; Subclass: Aspidochirotacea; Order: Aspidochirota; Genus: Holothuria Species: scabra

[0373] The material was collected from the shores of central West Coast of India during a low tide. The animals were brought to the laboratory and maintained in glass tanks containing seawater of salinity 30-32 per par (30%0) till further use.

Example 2

[0374] Extraction of the Dye

[0375] Extraction of the dye is a two step process. First an alcoholic extract is made and second the alcohol is evaporated and the fluorophore is separated and dissolved in solvent ether as follows:

[0376] Step 1: The animals were first washed with tap water and then with Milliq water (ultra pure water). The body was cut open with the sharp scissors and the female gonads were identified and removed. The color of the ovaries varied from yellow to orange. 1 milligram (1 mg) of the ovarian tissue is weighed and 70% ethyl alcohol was added to it in the ratio of 1:3 (weight by volume ratio). Yellowish orange colored pigment came out. The dye solution was decanted. To the remaining tissue again 70% ethyl alcohol was added and colored solution removed. These steps were repeated three times for extraction of the pigment without homogenizing the tissue of the ovary. The extract was filtered through Whattman No. 1 filter paper. The extract was was stores in a vial with screw cap at 4° C. and termed as “Stock dye solution”. The extract carrying both light- yellow and orange colored pigments from the ovarian tissue were characterized by the following methods. The properties were found the same. 1 mg ovarian tissue: 3 ml of 70% ethyl alcohol (3 times) i.e. 1 mg of pigment in 9 ml or 9000 microliter of alcohol.

[0377] Step 2: 15 ml of 70% alcoholic dye solution was taken, heated to coagulate the proteins and evaporated slowly to dryness in a water bath at 80 0centigrade. The dried material was not soluble in water and alcohol. It was soluble in ether. Ether was added to the dried material and allowed to stand at the room temperature for 15 minutes. The coloring portion got dissolved. It was then filtered by a Whattman no 1 filter paper to obtain a clear yellow colored solution, which was labeled as “Non-polar fluorescent dye”.

Example 3

[0378] Physical characteristics of the dye:

[0379] Color and solubility:

[0380] The said Dye solution is orange in color with the naked eye. In the daylight/tube light it gives varied colors emissions. The dye is insoluble in water but is soluble in non-polar solvent ether. Hence it is a non-polar dye. The dye has pH of 7.0.

Example 4

[0381] The said extract was subjected to paper electrophoresis. A whattman No.1 filter paper was soaked in phosphate buffer (0.1 M ) pH 7 and placed in an electrophoresis chamber. Both the electrodes were immersed in phosphate buffer. A spot was made with 5 microlitre ( μl) of the dye subjected to paper electrophoresis at 40 volts. The yellow spot does not move toward to any side. Further, it is getting dissolved only in the non-polar solvent ether. Since the dye is dissolving only in the ether, it is a non- polar dye.

Example 5

[0382] Photostability:

[0383] The ether extract of the dye is stable at room temperature. However to safeguard from evaporation the ether solution of the dye is kept at a cool place. Once the cells are labeled with the dye the fluorescence remains for months together at the room temperature.

Example 6

[0384] Test for electric charge of the Dye by electrophoresis:

[0385] The said extract was subjected to paper electrophoresis. A whattman No. 1 filter paper was soaked in phosphate buffer (0.1 M) pH 7 and placed in an electrophoresis chamber . Both the electrodes were immersed in phosphate buffer. A spot was made with 5 microlitre ( μl ) of the of the dye subjected to paper electrophoresis at 40 volts.

[0386] The yellow spot does not move toward to any side.

[0387] However, the 70% alcohol solution of isolation of phase 1 when tried, the colored spot moved towards positive pole. Which indicates that the non-polar pigment of the present dye in the phase 1 alcoholic extract of the ovarian tissue is attached to some negative protein. Chemical and HPLC analysis found the presence of proteins and carbohydrates in the alcoholic extract.

Example 7

[0388] Chemical Properties of Dye:

[0389] 1 ml of the dye solution, which is the alcoholic ovarian extract, was taken and 0.2 grams of di thio erythritol was incorporated. The solution does not get decolorized. It showed that the coloring part of the fluorescent dye does not contain a reducible group.

Example 8

[0390] Test of Non-proteinaceous Nature of the Dye.

[0391] 5 ml of the step 1 extract was subjected to heating in a water bath at 100 degree centigrade. Coagulation was observed in the alcoholic extract. This confirmed presence of protein in the starting alcoholic extract, however the protein was removed b coagulation. The colored solution came from the dried matter by dissolving it in ether. Since, the protein part has been removed the dye is said to be of non-proteinaceous nature.

Example 9

[0392] Absence of Agglutination of Bioactivity

[0393] One loop of live E. coli bacteria was placed in 50 microliter of water on a microscopic slide and mixed. To this was added 1microlitre (1 μl) of the dye solution. The alcohol was allowed to evaporate by leaving the slide on bench for 10-15Ssec. A coverslip was then placed on the preparation of bacteria and sealed. Similarly a control preparation of bacteria was made without any dye.

[0394] Both the slides were screened under oil immersion objective of an epifluorescence microscope (100× objective, 10× eye lens) for checking the agglutination bioactivity and the fluorescence under MIU, WB, WG and BF. It was seen that the bacteria did not show any clump formation (FIGS. 10, 10a).

[0395] The agglutination behavior was, however, noticed in the 70% alcoholic solution removed from the ovary tissue in phase 1 of isolation. But the protein was removed in the second phase of isolation and the present dye solution was of non-proteinaceous nature.

[0396] The absence of the tendency of cells to make aggregations in the presence of the present dye solution indicated that it does not contain a lectin like glycoprotein any more.

Example 10

[0397] Agglutination Bioactivity Test with Eukaryotic Sperms

[0398] One milliliter solution of oyster sperms (100 microliter) was placed in the cavity of a sedgewick counter used for phytoplankton counts. 1 microliter (1 μl) of dye solution was added. The slide was screened under 40× objective of a microscope with 10× eye lens. It was observed that the sperms did not make clumps, which confirmed lacking of lectin like activity of the dye.

Example 11

[0399] Test for Fluorophore and Protein Association in Phase 1 Material of 70% Alcoholic Solution but Absent in the Present Dye Phase 2 Isolation

[0400] As mentioned in examples 4and 6, the fluorophore shows non-polar behavior. But it appears that in the 70% alcoholic solution of ovarian tissue of isolation phase 1 the colored spot moves towards positive pole during paper electrophoresis. It may be bound in that phase to some component, which is negatively charged. The HPLC data of the 70% alcoholic solution detected only proteins and carbohydrates. As carbohydrates do not have an electric charge, it suggested that the colored component of the present dye in phase 1 (70% alcoholic solution) be attached to some protein, which is negatively charged. However, in isolation phase 2, which produces the present dye, it gets separated from the protein on coagulation by heating. It is the solution of phase 2 dye, which forms the present nonpolar dye solution.

Example 12

[0401] Method of Using the Non Polar Dye in Experiments

[0402] The dye is soluble in ether so a special method is adopted for of adding dye to the experimental animals. Live cells are taken on a clean slide. To this is added a measured amount of ultra pure/seawater depending upon the media in which the cells are cultured and remain alive in controls. The measured amount of the dye is placed on outer skirt of the drop and allowed to evaporate for 2-3 seconds. The solution is then mixed with tip of an eppendorf. A coverslip is immediately placed on the sample and sealed. The slides are labeled and the cells are viewed under an epifluorescence microscope.

Example 13

[0403] Nontoxicity Test of the Dye on Eukaryotic Cells

[0404] The extract was tested for nontoxicity upon the oyster sperms. The survival of the sperms in the experimental set ups was taken as a parameter for showing nontoxicity Male gonads of an oyster were removed and sperms were released in 100% seawater. These were filtered through a musline cloth to remove any debris. 1 mililiter (ml) of the sperm solution was taken and different concentrations (1 μl, 2 μl, 3 μl, 4 μl and 5 μl) of the Dye solutions were added. At every half an hour the observations of survival of the sperms were made under a microscope. The experiments were continued for 24 hours. The Controls were maintained without addition of Dye. It was seen that there was no harmful effect upon the survival of sperms with the addition of the dye. This proved nontoxic nature of the dye to estuarine and marine animal cells.

Example 14

[0405] Nontoxicity Test of the Dye on Prokaryoles

[0406] The extract was tested for toxicity upon gram negative E.coli bacteria by observing their survival/mortality. Live E.coli bacteria were taken on a clean slide. To this was added 25 μl ultra pure water. 1 μl of the dye is placed on outer skirt of the drop and allowed to evaporate for 2-3 seconds. The solution is then mixed with tip of an eppendorf. A cover slip is immediately placed on the sample and sealed. The slides are labeled and the cells are viewed under an epifluorescence microscope FIGS. 1,1a, 2 and 2a). The dead bacteria do not show any fluorescence (FIG. 3,3a).

Example 15

[0407] Dye Permeability Test in Oyster Eggs

[0408] The unfertilized, fertilized eggs and larvae of oysters were stained with the dye and egg suspension ratio of 1:50 microliter and screened under a fluorescent microscope. It was seen that the fluorescence was noticeable in the plasma membrane, nuclear envelop and chromatin. Though the wavelength ranges of emissions were the same and the colors were the hues of the same shade, there was a noticeable demarcation of boundaries of these parts of the cell (FIGS. 4,4a, 5,5a, and 6, 6a). This proved that the dye is permeable through the live and fixed cell membranes of egg plasma membrane, cytoplasm, nuclear membrane, nucleoplasm and chromatin. The absence of fluorescence of these parts of the cell in the dead cells showed that dye is impermeable to dead cell membranes.

Example 16

[0409] Detection of Live and Dead Bacteria in Cultures

[0410] A drop of the live E.coli bacteria in water (25 μl) was placed on a microscopic slide. To this was added 1 μl of the dye. The slide was sealed temporarily so as to save it from evaporation. It was seen under the microscope that the bacteria gave fluorescence in all the filter excitation ranges like WU, WB and WG. The controls were kept with the live E.coli without the stain and the dead E.coli. In both the controls the bacteria did not show fluorescence. This showed that the dye could be used for sorting live and dead bacteria in cultures (FIGS. 3,3a)

Example 17

[0411] Dye Useful for Checking Results of Cytotoxicity Bioassays

[0412] The quality of showing fluorescence in the live and fixed cells and not the dead cells is useful for checking cytotoxicity and antibacterial activity of various compounds by performing bioassays and checking the results with this dye.

Example 18

[0413] Dye Useful for Checking Bacterial Contamination

[0414] The quality of showing fluorescence in the live and fixed cells and not the dead cells is useful for checking contamination in the environmental and industrial samples where presence and absence of bacteria needs to be seen.

Example 19

[0415] Epifluorescence Microscopy of the Fluorophore

[0416] The epifluorescence microscopic studies are made by using this dye as a stain in the dilutions of 1:100 and recording emissions of light when excited by different cubes and compared the color hues with the known fluorochromes. It was noticed that the emissions were much stronger with the fluorophore though the shades of colors were the same. The screening was done using excitations of UV light and visible light spectra by WU, WB, WG and BF cubes of the Olympus reflected light. The details of cubes were as follows:

[0417] WU cube's wavelength range was 330 nm- 385 nm.

[0418] WB cube's wavelength range was 450 nm- 480 nm.

[0419] WG cube's wavelength range was 510 nm- 550 nm.

[0420] BF is for the bright field where an ordinary tungsten bulb delivers light.

Example 20

[0421] The emission ranges of the dye at different excitation ranges were found out. It was seen that excitation with the WU 330 nm-385 nm range emitted fluorescence in the 450 nm-470 nm range. Excitation with the WB filter having spectral range of 450 nm-480 nm emitted fluorescence in the 510 nm-570 nm range. The excitation with the WG filter having spectral range of 510 nm-550 nm emitted fluorescence in the 610nm-650 nm range. With BF the shades of yellowish grays were seen.

Example 21

[0422] Fluorescence Emissions in the Oyster Cells

[0423] The dye was used as microscopical stain on the dead, live and fixed eggs of the oyster. The slides were screened under an epifluorescent microscope. It was noticed that the dead cells do not take up dye and shows no fluorescence. (FIGS. 4, 4a, 5, 5a, 6, 6a, 7, 7a, 8 and 8a) .The fluorescence was observed in the eggs even 5 days after the first use of the dye and leaving the slides it the room temperature.

[0424] The live and fixed cells showed fluorescence. The excitation spectral range and the emitted fluorescence strictly followed the Stoke's law. These were different from the dye solution and was as given below:

[0425] Excitation with the WU 330 nm-385 nm range emitted fluorescence in the 470 nm-500 nm range. Excitation with the WB filter having spectral range of 450 nm-480 nm emitted fluorescence in the 570 nm-610 nm range. The excitation with the WG filter having spectral range of 510 nm-550 nm emitted fluorescence in the 610 nm-650 nm range. The epifluorescent microscopic screening of the dead, live and fixed eggs under Bright Field by using transmitted light emitted light in full white range of the visible spectra and depending upon the density of the cell ingredients gave hues of gray a phase contrast like effect.

Example 22

[0426] Fluorescence Emissions in the E.Coli Cells

[0427] The dye was used as microscopical stain on the E.coli. The slides were screened under a epi fluorescent microscope. It was noticed that the dead cells do not take up dye and show no fluorescence. (FIGS. 3, 3a). The live cells showed fluorescence. The excitation spectral range and the emitted fluorescence strictly followed the Stoke's law. These were different from the dye solution and was as given below:

[0428] Excitation with the WU 330 nm-385 nm range emitted fluorescence in the 470 nm-500 nm range. Excitation with the WB filter having spectral range of 450 nm-480 nm emitted fluorescence in the 570 nm-610 nm range. The excitation with the WG filter having spectral range of 510 nm-550 nm emitted fluorescence in the 610 nm-650 nm range. The control E. coli without dye did not show any fluorescence.

Example 23

[0429] Microphotography of the Fluorescent Cells with the Dye used as Stain

[0430] The microphotography of emitted fluorescence in the areas of slides without cells and with specimen cells, under WU 330 nm-385 nm range, WB 450 nm-480 nm range, WG 510 nm-550 nm range and Bright field was done by Kodak film of 400 ASA speed with an exposure varying from 50 to 60 seconds.

[0431] ADVANTAGES OVER THE PRESENTI MARKETED DYES

[0432] 1) The dye is non-radioactive as it is a dye from a natural source and not synthetic.

[0433] 2) The dye is nontoxic to estuarine, marine animals and gram negative E.coli bacteria

[0434] 3) The dye is cell membrane permeant and attaches itself to nuclear mebrane and chromatin also.

[0435] 4) The dye is permeant to cytoplasm and nucleoplasm.

[0436] 5) The dye is a non-polar fluorescent dye from a non-bioluminescent marine animal.

[0437] 6) The dye is a partially purified fluorophore in solution.

[0438] 7) This dye in its single form is equivalent to six synthetic fluorochromes which covers the major part of UV and visible light spectrum emission of fluorescent colors.

[0439] 8) The dye can be used as a quick microscopic stain giving a phase contrast effect without any extra expenses on phase contrast accessory of a microscope and without any lengthy protocols of fixations and preservations of specimens, especially on the spot quality check of live samples.

[0440] 9) Being non-degradable in quality of fluorescence at the room temperature, it does not require refrigeration while exporting the dye. The presently marketed fluorescent dyes are exported under refrigeration equivalent to −20 degree centigrade.

[0441] 10) Being non-degradable in quality of fluorescence on stained cells for longer duration, it does not require refrigeration while exporting stained slides. The dye solution can be marketed at 4 degree centigrade. The presently marketed fluorescent dyes exported under refrigeration equivalent to −20 degree centigrade.

[0442] 11) The dye has a pH in the range of 7.0 so in compositions the pH of the products will not change drastically.

[0443] 12) The fluorophore can be conjuagetd with other biomolecules.

[0444] 13) The dye is non-polar.

[0445] 14) It is soluble in ether

[0446] 15) The dye is non-degradable under natural conditions.

[0447] 16) The dye is non-degradable under natural conditions and also once it gets attached to the cell membranes. This can be of immense value to study live cell functions, organelle structure, cell sorting and flowcytometry.

[0448] 17) The dye solution is nontoxic to live bacteria and oyster eggs and sperms.

[0449] 18) The nontoxic nature of the dye has the advantage that this can be made a dye component of kits dealing with analysis of live cells

[0450] 19) The nontoxic nature of the dye has the advantage that this can be made a dye component of kits dealing in situ operational studies in oceanography.

[0451] 20) The dye does not stain and do not show fluorescence on dead bacteria. This is useful in tissue culture for checking live and dead cells.

[0452] 21) The dye being nontoxic to live cells can be useful in following up of cell lines.

[0453] 22) The dye is cell membrane permeant in the live and fixed cells. This is a natural dye so has the advantage over the synthetic ones because it will be more ecofriendly.

[0454] 23) The dye covers a wider range of emission wavelengths at selective excitations. This makes it a very acceptable component of dual emission dyes kits marketed presently for addition of a third emission color range.

[0455] 24) The dye does not undergo photobleaching while screening under the microscope.

[0456] 25) The dye does not show photobleaching once it stains the cells on slides. This makes it useful in histochemical studies.

[0457] 26) The dye emitted these fluorescence colors even at a dilution range of 1:400000 to 1:900000 times

[0458] 27) These multicolored emissions of the dye at different wavelengths of excitations are comparable to the fluorochrome microscopic stains already in the market.

[0459] 28) 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.

[0460] 29) The blue colored fluorescence of the present dye is also comparable to the emission of color by Hoechest 33258 used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.

[0461] 30) The blue colored fluorescence of the present dye is also comparable to the emission of color by Hoechest 33342 fluorochrome at the same wavelength excitation used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.

[0462] 31) The yellow colored fluorescence of the said dye in the visible range is comparable to the same colored emissions of Acridine orange used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.

[0463] 32) The yellow colored fluorescence of the said dye in the visible range is comparable to the same colored emissions of auramine used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.

[0464] 33) 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.

[0465] 34) The orange colored fluorescent emission is comparable to the orange fluorescence color of Propidiumr Iodide fluorochrome used as components of the non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.

[0466] 35) 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.

[0467] 36) 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.

[0468] 37) Unlike the synthetic commercial dyes used for the same purposes, the present dye is stable at the room temperature and has a long shelf life. Molecular non-radioactive kits of the said dye can be exported at the room temperatures.

[0469] 38) The said single dye has characteristics of atleast one hundred and twenty three different fluorochromes DAPI, Hoechest 33258, Hoechest 33342, FITC, acridine orange, auramine,Rhodamine, TRITC, and propidium iodide etc.) now in the market.

[0470] 39) Under ordinary light of microscope the hues of 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. The fluorescence color emissions follow Stoke's law of fluorescence.

[0471] 40) The microphotographs with Kodak film rolls show hues of the adjacent color emission wavelengths. Like when seen blue color fluorescence under the epifluorescence microscope in microphotograph the hues of green also comes.

[0472] 41) The microphotographs with Kodak film rolls show 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.

[0473] 42) The dye when seen orange fiuorescence color under the epifluorescence microscope in microphotograph the hues of red also come.

[0474] 43) The cytogenetic slides seen under all fluorescence gives a counterstain effect of cells with the background where no specimen but only dye is present.

[0475] 44) The dye can be used in fluorescent colors in variety of paints, inks, and textiles.

[0476] 45) The dye can be used in compositions of fluorescent dye for bleaching and brightening polymer.

[0477] 46) The dye can be used in leak detection with a full spectrum fluorescent dye.

[0478] 47) It can also be used in automated chemical metering system. It can also be used to mark the location of crashed aircraft, life crafts, and equipment for example rockets. Further it can be used in under sea probes.

[0479] 48) The nontoxic and cell permearit nature of the dye can be used as a component of the non-radioactive labeling and detection kits of biochemistry, cell biology, mmunochemistry, and molecular biology for labeling of DNA, RNA, Proteins and enzymes., Immunofluorescent detections, Counterstain of DIG-labeled oliogonucleotide probes and Anti-DIG Fab-fragments, Single and multiple cell quantitative fluorescence in Flowcytometry., Fluorochrome stains for epifluorescence microscopy.

[0480] 49) The dye can be used for a quick check of biocontamination in the health food industry, cosmetic industry, pharmaceutical and chemical industries, for rapid estimations of biocontaminants in laboratory cultures, for a rapid check of biopollutants under field conditions.

[0481] 50) The dye can be used in compositions where a nontoxic dye is required.

[0482] 51) The dye is environmentally friendly. It does not kill estuarine/marine animal larvae.

[0483] 52) The dye can be used natural colorant. A bioactive composition of the marine dye in the ratio of 1:40000 to 1:400000 to obtain fluorescence of six colors at three different wavelengths and a phase contrast effect under transmitted light.

[0484] 53) The dye being non-polar can easily be attached to variety of biomolecules.

[0485] 54) It fits in the criterion of a good dye demand of the industry as it shows the different parts of the cell at one emission and at different emissions.

[0486] References cited (Referenced By)

[0487] Bandaranayake, W. M. and Des Rocher,A 1999 (Marine biology 133;163-169)

[0488] Bitplane products at (http://www.bitplane.ch/public/support/standard/fluorochrome.htm)

[0489] Boehringer Mannheim GmbH, Biochemica, Catalogue , printed in Germany, Chapter II. Non-radioactive In Situ Hybridization Application Manual. 1992)

[0490] Chalfie M “Green fluorescent protein” in 1: Photochem Photobiol Oct 62 (4): 651-6 56 (1995)

[0491] Chalfie et al. SCIENCE 263 (1994) 802-805. Proteins with other wavelengths in the range of red and yellow are produced by induced mutations in some aminoacids.

[0492] Debra K. Hobson and David S. Wales.“Green dyes”, Journal of the Society of Dyers and Colourists (JSDC), 114, 42-44, (1998 ).

[0493] Fradkov A F, Chen Y, Ding L, Barsova E V, Matz M V,Lukyanov S A “Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far- red fluorescence. In 1:FEBS Lett Aug. 18: 479 (3): 127-30. ( 2000).

[0494] George L. Clark, “Pigments” in Encyclopaedia of chemistry, 2nd ed. Pages 833-835,1966.

[0495] Gurskaya N G, Fradkov A F, Terskikh A, Matz M V, Labas Y A, Martynov V I, Yanushevich Y G, Lukyanov K A, Lukyanov S A “GFP-like chromoproteins as a source of far-red fluorescent proteins” in 1: FEBS Lett Oct 19; 507 (1): 16-20 (2001).

[0496] Haughland R. P. Molecular probes. The Handbook of Fluorescent probes and Research Chemicals by Richard P. Haughland , 6th edition Printed in the united States of America, 1996.

[0497] R. Norman Jones in The encycopaedia of chemistry, 2nd edition,1966, Quote from pages 435- 436

[0498] Wachter R M, Elsliger M A, Kallio K, Hanson G T, Remington S J. In 1: Structure Oct. 15, 1998 ; 6(10): 1267-77 described “Structural basis of spectral shifts in the yellow-emission variants of green fluorescent protein”.

[0499] Youvan D C, Michel-Beyerie M E “Structure and fluorescence mechanism of GFP in National Biotechnology Oct.14, 1996 (10): 1219-20.

[0500] George L. Clark, “Fluorescence” in Encyclopaedia of chemistry, 2nd ed. Page 435-436, 1966.

[0501] Kowalzick L; Ott A; Waldmann T; Suckow M; Ponnighaus J, M. Vogtlandklinikum Plauen (Elsevier Science B.V2000).

[0502] Matz M V, Fradkov A F, Labas Y A, Savitsky A P, Zaraisky A G, Markelov M L, Lukyanov S A . “Fluorescent proteins from nonbioluminescent Anthozoa species ”,1: Nat Biotechnolo December 1999 : 17(10): 969-73.

[0503] Sepcic K, Turk T, Macek P “A fluorescent zoanthid pigment, parazoanthoxanthin A.” Toxicon, 36 (6): 937-940,1998.

[0504] Olympus Optical Co. Ltd, Tokyo Japan. Catalogue . “Instructions BX-FLA Reflected Light Fluorescence attachment” Page 16. 1999.

[0505] Shimomura,O, Johnson,F. H. and Saiga, Y “A novel reporter gene” Journal of cellular and comparative physiology, 59, 223-239, 1962.

[0506] Stainfile-Dyes A has given a Dye index of 264 dyes. Out of which 258 are synthetic and only six are natural dyes (http://members.pgonline.com/˜bryand/dyes/dyes.htm).

[0507] Wüerthner A. K. http://www.uniulm.de/uni/fak/natwis/oc2/ak wuert/publications.htm (referred from the internet).

[0508] Sigma-Aldrich “Biochemicals and reagents for life sciences research, 2000-2001 in their section immunochemicals of the catalogue under labeling reagents and cell linker labeling kits pp. 1454-1456. (web site www.sigma-aldrich.com)

[0509] US Patent Documents

44,063,878Dec. 20, 1977Weeks, B. W.4,452,822Jun. 5, 1984Shrikhande, Anil J4,774,339Sep. 27, 1988Haughland, R. P et al.4,762,7019 Aug. 1988Horan et al.4,783,4018 Nov. 1988Horan et al.4,859,58422 Aug. 1989Horan et al.5,248,782Sep. 28, 1993Haughland, R. P at al.5,187,288Feb. 16, 1993Kang, H. C. at al.5,274,113Dec. 28, 1993Kang, H. C et al5,321,26814 Jun. 1994Crosby David A and EkstromPhilip A5,304,493Apr. 19, 1994Nowak, A. V.5,405,416Apr. 11, 1995Swinton; Robert J,5,433,896Jul. 18, 1995Kang, H. C.5,451,663Sep. 19, 1995Kang, H. C5,770,205Jun. 23, 1998Collin, P. D5,710,197Jan. 20, 1998Fischer et al.5,876,762Mar. 2, 1999Collin, P. D5,985,330Nov. 16, 1999Collin, P. D5.858,761Jan. 12, 1999Tsubokura, et al.5,902,749May 11, 1999Lichtwardt et al.5,908,650Jun. 1, 1999Lenoble, et al5,920,429Jul., 6 1999Burns et al.5,935,808Aug. 10, 1999Hirschberg, et al5,989,135Nov. 23, 1999Welch; David Emanuel6,055,936May 2, 2000Collin; Peter Donald6,056,162May 2, 2000Leighley; Kenneth C.6,103,006Aug. 15, 2000DiPietro; Thomas C.6,110,566Aug. 29, 2000White et al.6,140,041Oct. 31, 2000LaClair; James J.6,165,384Dec. 26, 2000Cooper et al.6,180,154Jan. 30, 2001Wrolstad et al.International patents:EP020671830 Dec. 1986Cramer Randall JIE90137930 Jan. 1991Lee Linda G; MizePatrick DWO90100447 Jul. 1990.Swinton; Robert JDE 441647616 Nov. 1995S. Beckmann et al.WO 3067916 Nov. 1995S. Beckmann et al.DE 1961135122 Mar. 1996K-H. Etzbach et al.AU7041127 Oct. 1997Burns David M; PavelkaLee AWO 359262 Oct. 1997K.-H. Etzbach et al.DE 19648564 A128 May 1998C. Grund et al.DE 19650958 A110 Jun. 1998C. Grund et al.WO 98/236884 Jun. 1998C. Grund et al.DE 19643097 A123 Apr. 1998F. Würthner et al.,DE 19711445 A124 Sep. 1998F. Würthner et al.,WO 4158324 Sep. 1998F. Würthner et al.,DE1975564224 Jun. 1999Weimer Thomas DR.W0993891928 Sep. 1999Laclair James JWO00584065 Oct. 2000.Rosenblum Barnett B etal.WO993891615 Aug. 2000DiPietro; Thomas CWO992068829 Au. 2000Pavelka Lee et al.WO992068829 Aug. 2000White et al.

[0510]

5

TABLE 1

The Emissions of the different colored fluorescence of the fluorescent

dye when excited with different wavelength fluorescent filter cubes of

the Olympus epifluorescence microscope with dye solution and when

attached to cell membranes of the prokaryotic cells.

Name of the

fluorescent

Emitted

cube as

Emission

Color

given in the
Excitation

range of

of the

catalogue
range
Emission
dye when

dye when

of Olympus
of the
range
attached to
Emitted
attached to

Optical
fluores-
of dye
Cell mem-
Color of
Cell mem-

Co. Ltd.
cent cube
solution
branes
dye
branes

M WU
330-
450 nm-
470 nm-
Blue
Blue

385 nm
470
500

M WB
450-
510 nm-
570-
Green
Yellow

480 nm
570 nm
610 nm

MWG
510-
610 nm-
610 nm-
Orangish
Orangish

550 nm
650 nm
650 nm
Red
red

Bright field
Transmit-
White
White
Yellowish
Shades of

ted light
light
light
gray
yellowish

dark gray

[0511]

6

TABLE 2

The Emissions of the different colored fluorescence of the

fluorescent dye when excited with different wavelength

fluorescent filter cubes of the Olympus

epifluorescence microscope with dye solution and when attached

to cell membranes of the eukaryotic cells.

Name of the

Fluorescent

Emitted

cube as

Emission

Color

given in the
Excitation

range of

of the

catalogue
range
Emission
dye when

dye when

of Olympus
of the
range
attached to
Emitted
attached to

Optical
fluores-
of dye
Cell mem-
Color of
Cell mem-

Co. Ltd.
cent cube
solution
branes
dye
branes

M WU
330-
450 nm-
470 nm-
Blue
Blue

385 nm
470
500

M WB
450-
510 nm-
570-
Green
Yellow

480 nm
570 nm
610 nm

MWG
510-
610 nm-
610 nm-
Orangish
Orangish

550 nm
650 nm
650 nm
red
red

Bright field
Transmit-
White
White
Yellowish
Shades of

ted light
light
light
gray
yellowish

dark gray

[0512]

7

TABLE 3

Table showing various commercially important features of the three natural fluorescent dyes extracted from

the sea cucumber Holothuria scabra by the present inventors. (The features are kept under different

headings as shown in column no. 1)

Fluorescent Dye -(CSIR NF 140 2001)
Fluorescent Dye - (CSIR NF 152-2002)-
Fluorescent Dye - (CSIR NF 151-2002)

patent filed
Another co-pending Application
Present Application

Heading/title
U.S. patent Ser. No. 09/820,654
U.S. patent Ser. No.———
U.S. patent Ser. No.———

Name of
Sea cucumber
Sea cucumber
Sea cucumber

Animal/species

Holothuria scabra

Holothuria scabra

Holothuria scabra

(see example 1)
(see example 1)
(see example 1)

Bioactive
Pigment which is a fluorescent dye is
Pigment, which is a fluorescent dye, is
Pigment, which is a fluorescent dye, is

extract
extracted from skin of the animal with 50%
extracted from Live female gonads (ovarian
extracted from alcoholic extract of ovaries

alcohol
tissue) of the animal with 70% alcohol and
by heating it and then dissolving in the

(see example 2-5)
purified by filtration.
solvent either and purified by filtration.

(see example 2)
(see example 2)

Chemical nature
Non proteinaceous
Proteinaceous
Non proteinaceous and is non-polar.

(see example 7)
(see example 6, 8, 13)
(see example 2, 4, 8, 11)

Drug properties
In compositions of drugs of insecticidal,
Nontoxic to animal cells and bacteria.
Nontoxic to animal cells and bacteria.

pesticidal, veterinary medicines and
During bioassays the oyster sperms and
During bioassays the oyster sperms and

cosmetics for sunscreens.
bacteria remained alive in the sets treated
bacteria remained alive in the sets treated

(see examples 19-23)
with different concentrations.
with different concentrations.

The dye can be useful in drug delivery
The dye can be useful in drug delivery

studies for in situ applications
studies for in situ applications

(see examples 17, 18)
(see examples 13, 14)

Cell membrane
Stains and fluoresce live, dead, refrigerated
Stains and fluoresce live, and fixed cells. Do
Stains and fluoresce live, and fixed cells. Do

permeability
and fixed tissues.
not stain dead animal and bacterial cells.
not stain dead animal and bacterial cells.

Dye is cell membrane permeant
Dye is cell membrane permeant
Dye is cell membrane permeant

It is permeable to the nuclear membrane. It
It is permeable to the nuclear membrane. It
It is permeable to the nuclear membrane. It

also stains chromatin.
also stains chromatin.
also stains chromatin.

(see examples 21)
(see examples 24)
(see example 15)

Agglutination
Do not induce agglutination
Induces agglutination in oyster sperms, eggs
Neither induces agglutination in cells nor

induction

and bacterial cells but do not kill them.
kill them

(see examples 14, 15)
(See Examples 9, 10)

Effect on rate of
No effect on cell lines
Enhances rate of fertilization
No effect

fertilization of

(see examples 16)

oyster gametes

Biosurfactant
Biosurfactant nature present
Biosurfactant nature absent
Biosurfactant nature absent

nature
(see example 9)

Live/Dead
Stains and fluoresce all kind of cells and
Stains and fluoresce only live and fixed
Stains and fluoresce only live and fixed

bacterial
cellulose
cells. The dead bacteria do not stain and do
cells. The dead bacteria do not stain

contaminations
(see examples 12-17)
not fluoresce.
and do not fluoresce.

checks

(see examples 22)
(see examples 14, 16, 18)

Live/Dead
Stains and fluoresce all kind of cells and
Stains and fluoresce only live and fixed
Stains and fluoresce only live and fixed

animal cell
cellulose
cells. The dead animal cells do not stain and
cells. The dead animal cells do not stain

sorting in tissue
(see examples 12-17)
do not fluoresce.
and do not fluoresce.

cultures

(see examples 24)
(see examples 13, 15, 17)

Non-radioactive
Dye is non-radioactive as it is not a
Dye is non-radioactive as it is not a
Dye is non-radioactive as it is not a

dye
synthetic dye but is natural dye. Useful for
synthetic dye but is natural dye.
synthetic dye but is natural dye.

in situ non-radioactive kit compositions.
Dye is non-radioactive as it is not a
Dye is non-radioactive as it is not a

synthetic dye but is natural dye.
synthetic dye but is natural dye.

Photobleaching/
i.
Do not photobleach while screening
Do not photobleach while screening
Do not photobleach while screening

slides.

Photostability
ii.
Photostable at the room temperature
slides.
slides.

over one year
Photostable at 4 degree centigrade but
Photostable at the room temperature for

iii.
the fluorescence of the
after staining the cells, it remains non
several months.

dye does not change even when frozen at
degradable even at the room
The stained slides do not destain and

minus 20° C., a temperature at which
temperature for several months.
preserve fluorescence which enhances

the molecules are unable to attain the
The stained slides do not destain and
with maturity of slides.

energy necessary for activation like in
preserve fluorescence.
The fluorescence of the dye does not

extracts from luminescent organisms.
The fluorescence of the dye does not
change even when frozen at minus

(see example 18)
change even when frozen at minus
20° C., a temperature at which the

20° C., a temperature at which the
molecules are unable to attain the

molecules are unable to attain the
energy necessary for activation like in

energy necessary for activation like in
extracts from luminescent organisms.

extracts from luminescent organisms.
(see example 5)

(see examples 4, 25)

Quantum of

Very bright even at dilutions of 1:40000
Very bright even at dilutions of 1:400000
Higher than both the earlier dyes

fluorescence

(see examples 14)
and above
being much more brighter even at

(see examples 18, 19, 22)
dilutions of 1:400000-1:900000

and above.

(see examples 15, 16, 19)

Antimicrobial/

Antimicrobial/insecticidal/
Antimicrobial/insecticidal/
Antimicrobial/insecticidal/

insecticidal/

Pesticidal activities present.
Pesticidal activities absent.
Pesticidal activities absent.

Pesticidal

(see examples 10)
(see examples 17, 18)
(see examples 13, 14)

bioactivities

Emission ranges
1.
Dye emits three different colored
1.
dye emits six different colored
1.
dye emits six different colored

of dyes when

emissions

fluorescence at 3 different wavelengths

fluorescence at 3 different wavelengths

excited with
2.
Fluorescence blue color emission occur in

of the UV and visible ranges of the

of the UV and visible ranges of the

cubes of

the 380 nm-400 nm range of UVA when

fluorescent cubes of an epifluorence

fluorescent cubes of an epifluorescence

fluorescent

excited under ultra violet cube WU - 330

microscope,

microscope,

olympus BX-60

nm-385 nm excitation range,
2.
dye emits three different colored
2.
dye emits three different colored

microscope of
3.
Fluorescence yellow color emission

fluorescence at 3 different wavelengths

fluorescence at 3 different wavelengths

Olympus Co.

occurs in the 500 nm-570 nm range

of the UV and visible ranges of the

of the UV and visible ranges of the

Ltd Japan

when excited under WB cube of 450 nm-

fluorescent cubes of an epifluorescence

fluorescent cubes of an epifluorescence

having different

480 nm excitation range,

microscope when attached to the cell

microscope when attached to the cell

excitation
4.
fluorescence orange color emission

membranes. These three emission

membranes. These three emission

wavelength

occurs in the 570 nm-650 nm range

ranges are different from that of the dye

ranges are different from that of the dye

ranges:

when excited under WG cube of 510 nm-

alone.

alone.

WU range 330-

550 nm excitation range,
3.
Fluorescence blue color Emission occur
3.
fluorescence blue color emission occur

385 nm
5.
Phase contrast effect under bright field

in the 450 nm-470 nm range when dye

in the 450 nm-470 nm range when dye

WB range 450-

(see examples 14-17)

in solution is excited under ultra violet

in solution is excited under ultra violet

480

cube WU - 330 nm-385 nm excitation

cube WU - 330 nm-385 nm excitation

WG range 510-

range,

range,

550 nm

4.
fluorescence green color emission
4.
fluorescence green color emission

Bright field

occurs in the 510 nm-570 nm range

occurs in the 510 nm-570 nm range

when dye in solution is excited under

when dye in solution is excited under

WB cube of 450 nm-480 nm excitation

WB cube of 450 nm-480 nm excitation

range,

range,

5.
fluorescence orange color emission
5.
fluorescence orange color emission

occurs in the 610 nm-650 nm range

occurs in the 610 nm-650 nm range

when dye solution is excited under WG

when dye solution is excited under WG

cube of 510 nm-550 nm excitation

cube of 510 nm-550 nm excitation

range,

range,

6.
the dye emits hues of yellowish grays
6.
the dye emits hues of yellowish grays

under the ordinary transmitted light bulb

under the ordinary transmitted light bulb

of the epifluorescence microscope when

of the epifluorescence microscope when

seen under 100X objective,

seen under 100X objective,

7.
fluorescence blue color emission occur
7.
fluorescence blue color emission occur

in the 470 nm-500 nm range when dye

in the 470 nm-500 nm range when dye

attached to the cell membranes is

attached to the cell membranes is

excited under ultra violet cube WU -

excited under ultra violet cube WU -

330 nm-385 nm excitation range,

330 nm-385 nm excitation range,

8.
fluorescence green color emission
8.
fluorescence green color emission

occurs in the 570 nm-610 nm range

occurs in the 570 nm-610 nm range

when dye attached to the cell

when dye attached to the cell

membranes is excited under WB cube of

membranes is excited under WB cubes of

450 nm-480 nm excitation range,

450 nm-480 nm excitation range,

9.
fluorescence orange color emission
9.
ix. Fluorescence orange color emission

occurs in the 610 nm-650 nm range

occurs in the 610 nm-650 nm range

when dye attached to the cell

when dye attached to the cell

membranes is excited under WG cube

membranes is excited under WB cube

of 510 nm-550 nm excitation range,

of 510 nm-550 nm excitation range,

10.
the dye on cell membranes attached
10.
the dye on cell membranes attached

emits hues of yellowish grays under the

emits hues of yellowish grays under the

ordinary transmitted light bulb of the

ordinary transmitted light bulb of the

epifluorescence microscope when seen

epifluorescence microsope when seen

under 100X objective

under 100X objective

(see examples 19-23)

(see examples 19-22)

Other chemical
1.
decolorization by a reducing agent,
1.
is a non-reducible dye
1.
No decolorization by a reducing agent,

and physical
2.
not a synthetic compound,
2.
not a synthetic compound,
2.
not a synthetic compound,

Characteristics
3.
crude extract of the dye is yellowish
3.
crude extract of the dye is yelowish
3.
crude extract of the dye is orange in

of the dye

green in color,

orange in color,

color,

4.
purified dye being reddish brown colored
4.
partially purified dye being light
4.
The dye is in the solution form

powder when seen with the naked eye in

yellowish orange in color when seen
5.
The dye is extracted from alcoholic

the daylight,

with the naked eye in the daylight,

extract of the female gonads.

5.
Under tube light some hues of green are
5.
under tube light hues of multicolors are
6.
The alcoholic extract is heated and dried

emitted,

emitted,

at 80 degrees centigrade.

6.
amorphous in nature,
6.
cannot be made into powder
7.
The dried mass is dissolved in solvent

7.
soluble in water,
7.
is in a solution form

ether

8.
insoluble in the organic solvents like
8.
soluble in 70% ethyl alcohol, dilutions
8.
The dissolved dye pigment extract is

ethanol, methanol and acetone,

can be made in water/seawater.

filtered through whattman filer No. 1.

9.
is negatively charged,
9.
is negatively charged,
9.
The partially purified dye is also orange

10.
has a pH of 6.5,
10.
has a pH of 6.8-7.5,

in color.

11.
Presence of a phenolic group
11.
absence of a reducible group
10.
under tube light it emits variety of

12.
absence of a quinoid ring,
12.
proteinaceous in nature,

colors of the visible light spectrum at

13.
absence of a aromatic amine group
13.
The solvent front of TLC is 12.1 cm,

different angles.

14.
reducing sugar is absent,

four spots present.
11.
the colored pigment of the dye is

15.
pigment cum dye is a fluorescent dye
14.
Compound 1 spot has distance migrated

insoluble in water and alcohol.

and emits fluorescence when excited

of 11.1 cm. Its Rf value is 0.917.
12.
Tthe partially purified dye pigment is

with different wavelengths of UV and
15.
Compound 2 spot has distance migrated

soluble in solvent ether.

visible spectral ranges on a

of 9.9 cm. Its Rf value is 0.818.
13.
The further dilutions of the dye in

spectrophotometer,
16.
Compound 3 spot has distance migrated

experiments with animal and bacterial

16.
UV, visible spectroscopy from 300 nm-

of 8.0 cm. Its Rf value is 0.661.

cells in water and seawater can be made

700 nm and the peaks are marked at 379
17.
Compound 4 spot has distance migrated

by dissolving the ether extract of the dye

nm and 439 nm wavelengths,

of 5.6 cm. Its Rf value is 0.463

in different desired concentrations and

17.
UV, visible spectroscopy from 250 nm-
18.
It has carbohydrate

creating tribulations artificially.

350 nm and the peaks are at 272 nm
19.
It has protein
14.
The dye comes in contact with the cells

and 299 nm wavelengths,
20.
At least one of the proteins is a

and immediately permeates through the

18.
Fluorescent spectroscopy in the UV and

glycoprotein

membranes and stain them.

visible spectra, when excited with UV
21.
UV, visible spectroscopy the wavelength
15.
the dye has a pH of 7.0.

270 nm wavelength the fluorescence is

of excitation was maximum at 351 nm,
16.
there is absence of a reducible group.

emitted in the 324 nm-380 nm range

580 nm, 720 nm.

(see example 1-22)

which comes under the UVA wavelength
22.
the fluorescence emission spectrometric

range of ultraviolet rays of the sunlight,

analysis was in between 400 to 600 nm

19.
with excitation wavelength 450 nm in

when excited at 351 nm.

Fluorescent spectroscopy the
23.
The emission maxima were two peaks at

fluorescence emission occurred at

450 nm and 550 nm.

500 nm-580 nm with maximum

(see examples 1-25)

intensity,

20.
with excitation wavelength 540 nm in

Fluorescent spectroscopy, the

500 nm-620 nm with maximum intensity,

21.
with excitation wavelength 555 nm in

Fluorescent spectroscopy, the

fluorescence emission occurred at 575

nm-620 nm with maximum intensity,

22.
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 260 nm-280 nm

range emit bluish green hue color of

fluorescence.

(see examples 1-23)

Advantages
1.
dye is natural and not synthetic so steps
1.
dye is natural and not synthetic so steps
1.
dye is natural and not synthetic so

over presently

of harsh treatments with acids and

of harsh treatments with acids and

steps of harsh treatments with acids

marketed dyes

alkalies which are harmful to the

alkalies which are harmful to the

and alkalies which are harmful to

and common to

environment are not required. (see

environment are not required. (see

the environment are not required.

all the three yes

examples 1, 2)

examples 1, 2)

(see examples 1, 2)

2.
The dye fits in the criterion of a good
2.
The dye fits in the criterion of a good
2.
The dye fits in the criterion of a

dye according to which differentiation

dye according to which differentiation

good dye according to which

of various part of the cell can be done

of various part of the cell can be done

differentiation of various part of

under the same excitation range. (see

under the same excitation range. (see

the cell can be done under the

example 21)

example 24)

same excitation range. (see

example 15)

3.
The dye is nonradiactive as it is a dye
3.
The dye is nonradiactive as it is a dye
3.
The dye is nonradiactive as it is a dye

from a natural source and not synthetic.

from a natural source and not synthetic.

from a natural source and synthetic.

(see examples 1, 2)

(see example 1, 2)

(see examples 1, 2)

4.
the dye has antibacterial quality (see
4.
the dye is useful in drug industry
4.
the dye is useful in drug industry for

example 10)

making kits for checking bacterial

making kits for checking bacterial

5.
it has insecticidal qualities (see

contaminations. (see example 17, 18)

contaminations. (see example 13, 14)

example 19)
5.
the dye is useful in medical applications
5.
the dye is useful in medical applications

6.
the dye has pesticidal qualities. it kills

where fluorescence based in situ follow

where fluorescence based in situ

dog fleas and ticks (see example 23)

up of drugs or molecules need to be

follow up of drugs or molecules

7.
the dye is useful in drug industry for

seen since the dye is cell membrane

need to be seen since the dye is

making veterinary and insecticidal

permeant and nontoxic to cells. (see

cell membrane permeant and

compositions.

example 24)

nontoxic to cells. (see example 15)

8.
the dye is useful in medical applications
6.
This dye in its single form is equivalent
6.
This dye in its single form is equivalent

where X-ray defraction based studies

to six different synthetic fluorochromes

to six different synthetic fluorochromes

are needed. It can be a visual reality

giving same colored emission of

giving same colored emission of

without even exposing them on the the

fluorescent colors. While earlier

fluorescent colors. While earlier

X-ray films.

fluorochromes needs to be used in

fluorochromes needs to be used in mixed

9.
This dye in its single form is equivalent

mixed combinations this dye is a single

combinations this dye is a single dye

to three different synthetic

dye showing those effects. (see

showing those effects.
(see example 19-

fluorochromes giving same colored

example 19-23)

22)

emission of fluorescent colors. (see
7.
The dye can be used as a quick
7.
The dye can be used as a quick

example 14-17)

microscopic stain giving a phase

microscopic stain giving a phase contrast

10.
The dye can be used as a quick

contrast effect

effect without any extra expenses on

microscopic stain giving a phase

without any extra expenses on phase

phase contrast accessory of a

contrast effect without any extra

contrast accessory of a microscope and

microscope and without any lengthy

expenses on phase contrast accessory of

without any lengthy protocols of

protocols of fixations and preservations

a microscope and without any lengthy

fixations and preservations of

of specimens. Especially on the spot

protocols of fixations and preservations

specimens.Especially on the spot quality

quality check of live samples. (see

of specimens. Especially on the spot

check of live samples. (see example 19-

example 19-22)

quality check of live samples. (see

23)
8.
Being non degradable in quality of

example 21)
8.
Being non degradable in quality of

fluorescense for longer durations, it does

11.
Being non-degradable in quality of

fluorescence for longer durations, it

not require refrigeration while

fluorescence for longer durations, it

does not require refrigeration while

exporting. The presently marketed

does not require refrigeration while

exporting. The presently marketed

fluorescent dyes exported under

exporting. The presently marketed

fluorescent dyes exported under

refrigeration equivalent to −20 degree

fluorescent dyes exported under

refrigeration equivalent to −20 degree

centigrade. (see example 6)

refrigeration equivalent to −20 degree

centigrade. (see example 4, 25)
9.
Unlike the earlier known Green

centigrade. (see example 18)
9.
Unlike the earlier known Green

Fluorescent protein (GFP) from a marine

12.
Unlike the earlier known Green

Fluorescent protein (GFP) from a

jellyfish, the present dye is not a protein.

Fluorescent protein ( GFP) from a

marine jelly fish, our dye is though

The results are direct and 6 colored

marine jelly fish, our dye is not a protein

also is a protein but is not a reporter

emissions occurs at three excitation

and a reporter gene. The results are

gene. The results are direct and 6

wavelengths. (see example 2, 4, 8, 13)

direct. GFP absorbs blue light at 395 nm

colored emissions occurs at three
10.
the dye is soluble in solvent ether and is

and with a minor peak at 470 nm emits

excitation wavelengths. (see example

insoluble in water and alcohol. This

green light. Our dye emits 3 fluorescent

6, 8, 13)

gives its utility in specific dyes where

colors and at three different fluorescent
10.
The dye is soluble in 70% alcohol (see

non-polar dyes are needed. (see example

wavelengths. (see example 7)

example 2)

2)

13.
The dye is soluble in water so can be
11.
The dye is negatively charged protein
11.
The dye non-polar and has no charge.

used in components where water-

dye (see example 6, 8, 13)

(see example 2, 4, 8, 13)

soluble dyes are needed. The dye is
12.
The dye has a pH of 6.8-7.5 which is
12.
The dye has a pH of 7.0 and hence will

insoluble in the organic solvents like

almost neutral and hence will not effect

not effect final properties of pH in

ethanol, methanol and acetone.

final properties of pH drastically in

compositions. (see example 6)

(see example 2)

compositions. (see example 6)
13.
The dye is non-proteinaceous in nature

14.
The dye is negatively charged (see
13.
The dye is proteinaceous in nature and

so non-degradable under natural

example 8)

stable at 4 degree centigrade but once it

conditions. (see example 5)

15.
The dye has a pH of 6.5 which is almost

stains the cell membranes the
14.
The dye emmitted these fluorescence colors

neutral and hence will not effect final

fluorescence of cells is nondegradable

even at a dilution range of 1:450000

Properties of pH drastically in

for several months. (see example 4, 25)

times to 1:900000 times and above The

compositions. (see example 24)
14.
The dye emitted these fluorescence

fluorescence of the extract persisted even

16.
The dye is non-proteinaceous in nature so

colors even at a dilution range of

after at least 1 year at the room

non degradable under natural conditions.

1:400000 times and above The

temperature. These multicolored

(see example 18)

fluorescence of the extract persisted

emissions of the dye at different

17.
The dye has nature of a biosurfactant so

even after several months at the room

wavelengths of excitations are

can be used in soaps and toiletry

temperature. These multicolored

Comparable to the fluorochrome

compositions. (see example 9)

emissions of the dye at different

micorscopic stains already in the market.

18.
The dye has antimicrobial qualities (see

wavelengths of excitations are

(see example 19-23)

example 10)

comparable to the fluorochrome
15.
The blue colored fluorescence of the

19.
The dye emitted these fluorescence colors

microscopic stains already in the

present dye is comparable to the

even at a dilution range of 1:40000 times

market. (see example 18, 19, 22)

emission of same color by DAPI

(i.e. 1 gm powder of dye dissolved in 40
15.
The blue colored fluorescence of the

fluorochrome at the same wavelength

liters of ultrapure water). The

present dye is comparable to the

excitation, used as components of the

fluorescence of the extract persisted even

emission of same color by DAPI

non-radioactive labeling kits of

after at least 1 year at the room

fluorochrome at the same wavelength

biochemistry, cell biology,

temperature. These multicolored

excitation, used as components of the

immunochemistry, and molecurlar

emissions of the dye at different

non-radioactive labeling kits of

biology. (See example 19-22).

wavelengths of excitations are

biochemistry, cell biology,

16.
The blue colored fluorescence of the

comparable to the fluorochrome

immunochemistry, and molecular

present dye is also comparable to the

microscopic stains already in the market

biology. (see example 19-23)

emission of color by Hoechest 33258

(see example 14).
16.
The blue colored fluorescence of the

used as components of the non-

20.
The blue colored fluorescence of the

present dye is also comparable to the

radioactive labeling and detection kits of

present dye is comparable to the emission

emission of color by Hoechest 33258

biochemistry, cell biology,

of same color by DAPI fluorochrome at

used as components of the non-

immunochemistry, and molecular

the same wavelength excitation, used as

radioactive labeling and detection kits

biology. (see example 19-22).

components of the non-radioactive

of biochemistry, cell biology,
17.
The blue colored fluorescence of the

labeling kits of biochemistry, cell

immunochemistry, and molecular

present dye is also comparable to the

biology, Immunochemistry, and

biology. (see example 19-23)

emission of color by Hoechest 33342

molecular biology. (see example 14-17)
17.
The blue colored fluorescence of the

fluorochrome at the same wavelength

21.
The blue colored fluorescence of the

present dye is also comparable to the

excitation used as the same wavelength

present dye is also comparable to the

emission of color by Hoechest 33342

non-radioactive labeling and detection

emission of color by Hoechest 33258

fluorochrome at the same wavelength

kits of biochemistry, cell biology,

used as components of the non-

excitation used as components of the

immunochemistry, and molecular

radioactive labeling and detection kits of

non-radioactive labeling and detection

biology, (see example 19-22).

biochemistry, cell biology,

kits of biochemistry, cell biology,
18.
The yellow colored fluorescence of the

immunochemistry, and molecular

immunochemistry, and molecular

said dye in the visible range is

biology. (see example 14-17)

biology. (see example 19-23)

comparable to the same colored

22.
The blue colored fluorescence of the
18.
The yellow colored fluorescence of the

emissions of Acridine orange used as

present dye is also comparable to the

said dye in the visible range is

components of the non-radioactive

emission of color by Hoechest 33342

comparable to the same colored

labeling and detection kits of

fluorochrome at the same wavelength

emissions of Acridine orange used as

biochemistry, cell biology,

excitation used as components of the

components of the non-radioactive

immunochemistry, and molecular

non-radioactive labeling and detection

labeling and detection kits of

biology, (see example 19-22).

kits of biochemistry, cell biology,

biochemistry, cell biology,
19.
The yellow colored fluorescence of the

immunochemistry, and molecular

immunochemistry, and molecular

said dye in the visible range is

biology.(see example 14-17)

biology. (see example 19-23

comparable to the same colored

23.
The yellow colored fluorescence of the
19.
The yellow colored fluorescence of the

emissions of auramine used as

said dye in the visible range is

said dye in the visible range is

components of the non-radioactive

comparable to the same colored

comparable to the same colored

labeling and detection kits of

emissions of Acridine orange used as

emissions of auramine used as

biochemistry, cell biology,

components of the

components of the non-radioactive

immunochemistry, and molecular

Non-radioactive labeling and detection

labeling and detection kits of

biology. (see example 19-22).

kits of biochemistry, cell biology,

biochemistry, cell biology,
20.
The yellow colored fluorescence of

Immunochemistry, and molecular

immunochemistry, and molecular

the said dye in the visible range is

biology.. (see example 14-17)

biology. (see example 19-23)

comparable to the same colored

24.
The yellow colored fluorescence of the
20.
The yellow colored fluorescence of the

emissions of FITC used as components

said dye in the visible range is

said dye in the visible range is

of the non-radioactive labeling and

comparable to the same colored

comparable to the same colored

detection kits of biochemistry, cell

emissions of auramine used as

emissions of FITC used as components

biology., immunochemistry, and

components of the non-radioactive

of the non-radioactive labeling and

molecular biology. (see example 19-22).

labeling and detection kits of

detection kits of biochemistry, cell
21.
The orange colored fluorescent emission

biochemistry, cell biology,

biology, immunochemistry, and

is comparable to the orange

immunochemistry, and molecular

molecular biology. (see example 19-23)

fluorescence Color of Propidium Iodide

biology.. (see example 14-17)
21.
The orange colored fluorescent emission

fluorochrome used as components of the

25.
The yellow colored fluorescence of the

is comparable to the orange fluorescence

non-radioactive labeling and detection

said dye in the visible range is

color of Propidium Iodide fluorochrome

kits of biochemistry, cell biology,

comparable

used as components of the non-

immunochemistry, and molecular

To the same colored emissions of FITC

radioactive labeling and detection kits

biology. (see example 19-22).

used as components of the non-

of biochemistry, cell biology
22.
The orange colored fluorescent emission

radioactive

immunochemistry, and molecular

is comparable to the orange

Labeling and detection kits of

biology. (see example 19-23)

fluorescence Color of Rhodamine

biochemistry, cell biology,
22.
The orange colored fluorescent emission

non-radioactive Labeling and detection

immunochemistry, and

is comparable to the orange

kits of biochemistry, cell biology,

Molecular biology.. (see examples 14-

fluorescence color of Rhodamine

immunochemistry, and molecular

17)

fluorochrome used as components of the

biology. (see example 19-22).

26.
The orange colored fluorescent emission

non-radioactive labeling and detection
23.
The orange colored fluorescent emission

is comparable to the orange fluorescence

kits of biochemistry, cell biology,

is comparable to the ornage

color of Propidium Iodide fluorochrome

immunochemistry, and molecular

fluorescence color of TRITC

used as components of the non-

biology. (see example 19-23)

fluorochrome used as components of the

radioactive
23.
The orange colored fluorescent emission

non-radioactive labeling and detection

labeling and detection kits of

is comparable to the orange fluorescence

kits of biochemistry, cell boilogy,

biochemistry, cell biology,

color of TRITC fluorochrome used as

immunochemistry, and molecular

immunochemistry, and

components of the non-radioactive

biology.. (see example 19-22).

molecular biology.. (see example 14-

labeling and detection kits of
24.
Unlike the synthetic commercial dyes

17)

biochemistry, cell biology,

used for the same purposes, the present

27.
The orange colored fluorescent emission

immunochemistry, and molecular

dye is stable at the room temperature

is comparable to the orange fluorescence

biology. (see example 19-23)

and has a long shelf life. Molecular non-

color of Rhodamine fluorochrome used as
24.
Unlike the synthetic commercial dyes

radioactive kits of the said dye can

components of the non-radioactive

used for the same purposes, the present

be exported at the room temperatures..

labeling and detection kits of

dye is stable at the room temperature

(see example 19-22).

biochemistry, cell biology,

and has a long shelf life. Molecular non-
25.
The said single dye has characteristics

immunochemistry, and

radioactive kits of the said dye can be

of at least one hundred and twenty three

molecular biology.. (see example 14-

exported at the room temperatures. (see

different fluorochromes (DAPI,

17)

example 19-23)

Hoechest 33258, Hoechest 33342,

28.
The orange colored fluorescent emission
25.
The said single dye has characteristics

FITC, acridine orange,

is comparable to the orange fluorescence

of at least one hundred and twenty three

auramine, Rhodamine, TRITC, and

color of TRITC fluorochrome used as

different fluorochromes (DAPI,

propidium iodide etc.) now in the

components of the non-radioactive

Hoechest 33258, Hoechest 33342,

market. (see example 19-22).

labeling

FITC, acridine orange,
26.
Under ordinary light of microscope

and detection kits of biochemistry, cell

auramine, Rhodamine, TRITC, and

the hues of grays produce a phase

biology, immunochemistry, and

propidium iodide etc.) now in the

contrast effect which is useful in

molecular

market. (see example 19-23)

rapid screening of cytogentical,

biology.. (see example 14-17)
26.
under ordinary light of microscope

cytological, and histochemical slides

29.
Unlike the synthetic commercial dyes

hues of grays produce a phase contrast

and save expenses on the extra

used for the same purposes, the present

effect which is useful in rapid screening

phase contrast accessory

component of microscope. The

dye is stable at the room temperature and

of cytogentical, cytological, and

fluorescence color emissions follow

has a long shelf life. Molecular non-

histochemical slides and save expenses

Stoke's law of fluorescence.. (see

radioactive kits of the said dye can be

on the extra phase contrast accessory

example 19-22).

exported at the room temperatures.. (see

component of microscope. The
27.
The microphotograhs with Kodak film

example 18).

fluorescence color emissions follow

rolls show hues of the adjacent color

30.
The said single dye has characteristics of

Stoke's law of fluorescence. (see

emission wavelengths. Like when seen

at least one hundred and twenty three

example 19-23)

blue color fluorescence under the

different fluorochromes (DAPI, Hoechest
27.
The microphotographs with Kodak film

epifluorescence microscope in

33258, Hoechest 33342, FITC, acridine

rolls shows hues of the adjacent color

microphotograph the hues of green also

orange, auramine, Rhodamine, TRITC,

emission wavelengths. Like when seen

comes.. (see example 19-22).

and propidium iodide etc.) now in the

blue color fluorescence under the
28.
The microphotographs with Kodak film

market.

epifluorescence microscope in

rolls shows hues of the adjacent color

31.
under ordinary light of microscope the

microphotograph the hues of green also

Emission wavelengths. Like when seen

hues of grays produce a phase contrast

comes. (see example 19-23)

yellow color fluoroscence under the

effect which is useful in rapid screening
28.
The microphotographs with Kodak film

epifluorescence microscope in

of cytogentical, cytological, and

rolls shows hues of the adjacent color

microphotograph the hues of green also

histochemical slides and save expenses

emission wavelengths. Like when seen

comes. The dye when seen orange

on the extra phase contrast accessory

yellow color fluorescence under the

fluorescence color under the

component of microscope. The

epifluorescence microscope in

epifluorescence microscope in

fluorescence color emissions follow

microphotograph the hues of red also

Microphotograph the hues of red also

Stoke's law of fluorescence.

comes. The dye when seen orange

comes.. (see example 19-22).

32.
The microphotographs with Kodak film

fluorescence color under the
29.
The cytogenetic slides seen under all

rolls shows hues of the adjacent color

epifluorescence microscope in

fluorescence gives a counterstain effect

emission wavelengths. Like when seen

microphotograph the hues of red also

of cells with the background where no

blue color fluorescence under the

comes. (see example 19-23)

specimen but only dye is present.. (see

epifluorescence microscope in
29.
The cytogenetic slides seen under all

example 19-22).

microphotograph the hues of green also

fluorescence gives a counterstain effect
30.
The dye can be used for the preparation

comes.

of cells with the background where no

of polyvinyl chloride film that exhibits

33.
The microphotographs with Kodak film

specimen but only dye is present. (see

fluorescent colors. It also can be used in

rolls shows hues of the adjacent color

example 19-23)

fluorescent colors in variety of paints,

emission wavelengths. Like when seen
30.
The dye can be used for the preparation

inks and textiles.

yellow color fluorescence under the

of polyvinyl chloride film that exhibits
31.
The dye can be used in compositions of

epifluorescence microscope in

fluorescent colors. It also can be used in

fluorescent dye for bleaching and

microphotograph the hues of green also

fluorescent colors in variety of paints,

brightening polymer. The dye can be

comes. The dye when seen orange

inks, textiles.

used in leak detection with a full

fluorescence color under the
31.
The dye can be used in compositions of

spectrum fluorescent dye. It can also be

epifluorescence microscope in

fluorescent dye for bleaching and

used in automated chemical metering

microphotograph the hues of red also

brightening polymer. The dye can be

system. It can also be used to mark the

comes.

used in leak detection with a full

location of crashed aircrafts, life crafts,

34.
The cytogenetic slides seen under all

spectrum fluorescent dye. It can also be

and equipment for example rockets.

fluorescence gives a counterstain effect of

used in automated chemical metering

Furthur it can be used in under sea

cells with the background where no

system. It can also be used to mark the

probes. The dye can be used in photo

specimen but only dye is present.

location of crashed aircrafts, life crafts,

chemotherapy of skin cancers.

35.
The dye can be used for the preparation

and equipment for example rockets.
32.
It can be used as fluorescent in situ

of polyvinyl chloride film that exhibits

Further it can be used in under sea

hybridization application kit component

fluorescent colors. It also can be used in

probes. The dye can be used in photo

for =molecular diagnostics. It can also be

fluorescent colors in variety of paints,

chemotherapy of skin cancers.

used as a component of the non-

inks, textiles.
32.
It can be used as fluorescent in situ

radioactive labeling and detection kits of

36.
The dye can be used in compositions of

hybridization application kit component

biochemistry, cell biology,

fluorescent dye for bleaching and

for molecular diagnostics. It can also be

immunochemistry, and molecular

brightening polymer. The dye can be used

used as a component of the non-

biology for labeling of DNA, RNA,

in leak detection with a full spectrum

radioactive labeling and detection kits of

Proteins and enymes.,

fluorescent dye. It can also be used in

biochemistry, cell biology,

Immunofluorescent detections,

automated chemical metering system. It

immunochemistry, and molecular

Counterstain of DIG-labeled

can also be used to mark the location of

biology for labeling of DNA, RNA,

oliogonucleotide probes and Anti-DIG

crashed aircrafts, life crafts, and

Proteins and enzymes,

Fab-fragments, Single and multiple cell

equipment for example rockets. Further it

Immunofluorescent detections,

quantitative fluorescence in

can be used in under sea probes. The dye

Counterstain of DIG-labeled

Flowcytometry., Fluorochrome stains for

can be used in photo chemotherapy of

oliogonucleotide probes and Anti-DIG

epifluorescence microscopy

skin cancers.

Fab-fragments, Single and multiple cell
33.
The dye can be used for a quick check of

37.
The dye can be used as chromatophore

quantitative fluorescence in

biocontamination in the health food

sunscreen component of cosmetics

Flowcytometry., Fluorochrome stains

industry, cosmetic industry,

creams and lotions.

for epifluorescence microscopy.

pharmaceutical and chemical industries,

38.
The water miscible quality of the dye can
33.
The dye can be used for a quick check of

for rapid estimations of biocontaminants

make it easily miscible in moisturizers. It

biocontamination in the health

in laboratory cultures, for a rapid check

can be used as fluorescent in situ

food industry, cosmetic industry,

of biopollutants under field conditions

hybridization application kit component

pharmaceutical and chemical industries,
34.
The dye can be used natural colorant

for molecular diagnostics. It can also be

for rapid estimations of

35.
The dye being a non-polar fluorochrome,

used as a component of the non-

biocontaminants in laboratory cultures,

which is separated from the protein part,

radioactive labeling and detection kits of

for a rapid check of biopollutants under

can be used in formulations of variety of

biochemistry, cell biology,

field conditions (see example 24)

new molecules by attachments.. (see

immunochemistry, and molecular
34.
The dye can be used natural colorant

example 2, 4, 8, 13)

biology for labeling of DNA, RNA,
35.
Dye can be used in fertilization rate

Proteins and enzymes,

enhancements in medical, biomedical,

Immunofluorescent detections,

agricultural and aquaculture science (see

Counterstain of DIG-labeled

example 14, 15)

oliogonucleotide

probes and Anti-DIG Fab-fragments,

Single and multiple cell quantitative

fluorescence in Flowcytometry.,

Fluorochrome stains for epifluorescence

microscopy.

39.
The dye can be used for a quick check of

biocontamination in the health food

industry, cosmetic industry,

for rapid estimations of biocontaminants

in laboratory cultures, for a rapid check

of biopollutants under field conditions. It

can also be a competitive inhibitor of

cholinesterases.

40.
The dye can be used in anti microbial

compositions.

41.
The dye can be used as a biosurfactant in

toiletry compositions

42.
The dye can be used natural colorant

Specific uses of
a.
preparation of flexible polyvinyl
a.
use of fluorescent colors in variety of
a.
use of fluorescent colors in variety

the 3 natural

chloride film that exhibits

paints, inks, textiles;

of paints, inks, textiles;

fluorescent dyes

fluorescent colors;

described here.
b.
.use of fluorescent colors in variety of
b.
a composition of fluorescent dye for
b.
a composition of fluorescent dye for

paints, inks, textiles;

bleaching and brightening polymer;

bleaching and brightening polymer;

c.
a composition of fluorescent dye for
c.
leak detection with a full spectrum
c.
leak detection with a full spectrum

bleaching and brightening polymer;

fluorescent dye;

fluorescent dye;

d.
leak detection with a full spectrum
d.
use in automated chemical metering
d.
use in automated chemical metering

fluorescent dye;

system;

system;

e.
use in automated chemical metering
e.
to mark location of crashed air-crafts,
e.
to mark location of crashed

system;

life crafts, and equipment for example

air-crafts, life crafts, and

f.
.to mark location of crashed air-crafts,

rockets;

equipment for example rockets;

life crafts, and equipment for example
f.
under sea probes;
f.
under sea probes;

rockets;
g.
fluorescent in situ hybridization
g.
chromatophore sunscreen component of

g.
under sea probes;

application kit component for molecular

cosmetics creams and lotions;

h.
UVA is used in photo chemotherapy of

diagnostics;
h.
fluorescent in situ hybridization

skin cancers;. (see example 11)
h.
component of the non-radioactive

application kit component for molecular

i.
Chromatophore sunscreen component of

labeling and detection kits of

diagnostics;

cosmetics creams and lotions;

biochemistry, cell molecular
i.
component of the non-radioactive

j.
the water miscible quality of the dye can

immunochemistry, and molecular

labeling and detection kits of

make it easily miscible in moisturizers;

biology for labeling of DNA, RNA,

biochemistry, cell biology,

k.
fluorescent in situ hybridization

Proteins and enzymes;

immunochemistry, and molecular

application kit component for molecular
i.
immunofluorescent detections;

biology for labeling of DNA, RNA,

diagnostics;
j.
counterstain of DIG-labeled

Proteins and enzymes;

l.
component of the non-radioactive

oligonucleotide probes and Anti-DIG
j.
immunofluorescent detections;

labeling and detection kits of

Fab-fragments;

biochemistry, cell biology,
k.
single and multiple flow cytometry
k.
counterstain of DIG-labeled

immunochemistry, and molecular

applications;

oligonucleotide probes and Anti-DIG

biology for labeling of DNA, RNA,
l.
fluorochrome stains for epifluorescence

Fab-fragments;

Proteins and enzymes;

microscopy;. (see example 19-23).
l.
singel and multiple flow cytometry

m.
immunofluorescent detections;
m.
for a quick check of biocontamination

applications;

n.
counterstain of DIG-labeled

in the health food industry, cosmetic
m.
fluorochrome stains for epifluorescence

oligonucleotide probes and Anti-DIG

industry, pharmaceutical and chemical

microscopy;. (see example 19-22).

Fab-fragments;

industries;. (see example 22).
n.
for a quick check of biocontamination in

o.
single and multiple flow cytometry
n.
for rapid estimations of biocontaminants

the health food industry, cosmetic

applications;

in laboratory cultures;. (see example

industry, pharmaceutical and chemical

p.
fluorochrome stains for epifluorescence

22)

industries;. (see example 14, 16, 18)

microscopy;
o.
for a rapid check of biopollutants under
o.
For rapid estimations of biocontaminants

q.
for a quick of biocontamination in

field conditions;. (see example 22).

in laboratory cultures. (see example

the health food industry, cosmetic
p.
agglutination compositions;. (see

14, 16 and 18)

industry, pharmaceutical and chemical

example 14, 15)
p.
for a rapid check of biopollutants under

industries;
q.
a natural colorant;

field conditions; (see example 14, 16, 18)

r.
for rapid estimations of biocontaminants
r.
a bioactive composition of the marine
q.
for a rapid check of dead and live

in laboratory cultures;

dye in the ratio of 1:400000 in ultrapure

bacteria E. coli.. (see example

s.
for a rapid check of biopollutants under

water to obtain fluorescences of six

14, 16, 18)

field conditions;

colors at six different wavelengths and a
r.
a natural colorant; a bioactive

t.
a competitive inhibitor of

phase contrast effect under transmitted

composition of the marine dye in the

cholinesterases;

light;. (see example 18, 19, 22)

ratio of 1:400000 to obtain

u.
in antimicrobial compositions;. (see
s.
a dye for various fluorescent

fluorescences of six colors at three

example 10)

applications to be performed in areas of

different wavelengths and a

v.
as a biosurfactant in toiletry

sub zero temperatures.

phase contrast effect under transmitted

compositions;. (see example 9)
t.
for fertilization rate increase in

light;. (see example 13, 14)

w.
a natural colorant;

aquaculture industry. (see example
s.
a dye for various fluroescent applications

x.
a bioactive composition of the marine

14, 15)

to be performed in areas of sub zero

dye in the ratio of 1:40000 in ultrapure
u.
for cell permeant membrane dye

temperature.. (see example 5)

water to obtain fluorescences of three

compositions.. (see example 24)
t.
a dye for making conjugants with variety

colors at three different wavelengths and
v.
for identification of dead and live cells

of biomolecules.

a phase

in tissue cultures.. (see example 24)
u.
for cell permeant membrane dye

contrast effect under transmitted light;
w.
for identification of dead and live cells/

compositions.

(see example 14)

bacteria in food industries. (see
v.
for identification of dead and live cells in

y.
a dye br various fluorescent

example 24)

tissue cultures.. (see example 13, 15, 17)

applications to be performed in areas of
x.
for dye compositions in biosensors
w.
for dye compositions in biosensors

sub zero temperatures.. (see example
y.
as dye composition in molecular and
x.
as dye composition in molecular and

18)

microbiological kits.. (see example

microbiological kits. (see example

24)

13, 15, 17)

y.
A non-polar dye for detection of lipids

and oils.. (see example 4, 8, 11)

	Number	Date	Country
Parent	09820654	Mar 2001	US
Child	10107588	Mar 2002	US

Natural non-polar fluorescent dye from a non-bioluminescent marine invertebrate, compositions containing the said dye and its uses

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

FIELD OF THE INVENTION

Continuation in Parts (1)