GB2373788A - Natural fluorescent dye extract from the invertebrate marine sea cucumber Holothuria scabra - Google Patents

Abstract

The present invention discloses the process of extraction, purification and characterization of a fluorescent dye from a marine echinoderm <I>Holothuria scabra</I> (a marine sea cucumber), compositions containing the dye and various applications of the dye. Bioactive extracts obtained from any marine organism and useful as a natural fluorescent dye are also claimed provided they share the 34 characteristics set out in claim 1 with the <I>Holthuria scabra</I> extract. The dye is non-proteinaceous and emits three different coloured fluorescences at three different excitation wavelengths. A wide range of uses are claimed.

Description

NATURAL FLUORESCENT DYE FROM A MARINE INVERTEBRATE The present invention relates to a novel fluorescent dye obtained from a marine animal invertebrate, Holothuria scabra. The invention also provides a process for the extraction, purification and characterization of this new dye, which is a natural dye from marine invertebrate, especially sea cucumber. Sea cucumbers are echinoderms, members of the group of spiny skinned animals that also includes starfishes and sea urchins. They have the following taxonomic position.

The said sea cucumber has following taxonomic position: Subkingdom: Metazoa Phylum: Echinodermata Sub-Phylum: Eleutherozoa Class: Holothuroidea Subclass : Aspidochirotacea, Dendrochirotacea, Apodacea Order: Dendrochirota, Aspidochirota, Elasipoda Molpadonia and Apoda Amongst these orders, sea cucumber Holothuria scabra belongs to: Order: Aspidochirota Family: Holothuriidae Genus: Holothuria Species: scabra Echinoderms are coelomate invertebrates which are exclusively marine, never colonial, are unsegmented with a basic pentameric radial, have symmetry in the adult form, no head or brain, and are distinguished from all other animals by structural peculiarities of the skeleton and coelom. Class Holothuroidea has animals with body bilaterally symmetrical, usually elongated in the oral-aboral axis, having mouth at or near one end and anus at or near the other end. The body surface IS coarse, the endoskeleton reduced to microscopic spicules or plates embedded in the body wall, the mouth surrounded by a set

of tentacles attached to water vascular system ; podia or tube feet are usually present and locomotory ; the alimentary canal is long and coiled, and cloaca usually with respiratory trees; sexes are usually separate and the gonad single or paired tuft of tubules. They are sedentary forms either attached to hard substrate or burrowed into soft sediments with anterior and posterior ends projected. They occur in all seas chiefly in shallow waters, a few species occur in depths greater than 1000 meters. The species Holothuria sabra also called by some as Metriatyla sabra 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.

Pigments can be divided into inorganic and organic categories. The former are the inorganic chemical 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 Brazil 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). Debra K. Hobson and 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. Stainsfile-Dyes A gives Dye Index of 264 dyes. Out of which only six are natural dyes from all types of living organisms http://members. pgonline. com/-bryand/dyes/dyes. htm Recently, several patents have been published about natural dyes but majority of them are from plants. Wrolstad, et al. Described natural colorant from potato extract (US Patent No. 6,180, 154 published on January 30,2001). Shrikhande, Anil J in US patent No. 4,452, 822 published on June 5,1984 reported extraction and intensification of anthocyanins from grape pomace and other material. Lenoble, et at. US patent No. 5,908, 650 released on June 1, 1999 described a new composition to enhance the red colour of anthocyanin pigment.

Carotenoid-producing bacterial species are disclosed in two US patents Number 5,935, 808 published on August 10,1999, inventors Hirschberg, et al. and 5.858, 761

January 12, 1999 inventors Tsubokura, et al. Collin ; Peter Donald in his US patent No. 6, 055, 936 published on May2, 2000 disclosed sea cucumber carotenoid lipid fractions and process.

All these colorants and dyes are however not fluorescent. Fluorescent dyes most of which are synthetic are disclosed in several US and International patents. These have been used in variety of applications. The number 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 (US patent No.

5,989, 135 released on date November 23,1999 disclosed a luminescent golf ball. White et al. (US patent No. 6,110, 566, dated August 29,2000 and W09920688) described a flexible polyvinyl chloride film that exhibits durable fluorescent colours.

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 30 December 1986) described a composition with fluorescent dye for bleaching and brightening of polymer.

Leak detection is another utility disclosed by some (Leighley, Kenneth C. US patent 6,056, 162 dated 2 may, 2000). Cooper et al. US patent 6,165, 384 published on December 26 2000 disclosed a full spectrum fluorescent dye composition for the same purposes.

Lichtwardt et al. US patent 5,902749 dated May 11,1999 use fluorescent dye in an automated chemical metering system.

The reports from marine animals are few. A green fluorescent protein GFP-a novel reporter gene has been described from the pacific jellyfish, Aequora aequora by Shimomura, 0, Johnson, F. H. and Saiga, Y (Journal of cellular and comparative physiology, 59,223-239, 1962). GPF is characterized by the presence of highly fluorescent chromatophore. Purified GFP absorbs blue light maximally at 395 nm with a minor peak at 470 nm and emits green light. Sepcic K, Turk T, Macek P reported a fluorescent zoanthid pigment, parazoanthoxanthin A. Toxlcon, 36 (6): 937-940,1998.

Manne dyes have several uses as dye on its own and as a part of compositions.

Several authors have disclosed fluorescent dye blends for multiple purposes (Burns, et al. In US patent No. 5, 920, 429 published on July 6, 1999 ; Bums ; David M and Pavelka Lee A, (International patent AU704112). The marine dye compositions have been used in a number of applications to mark the location of crashed aircraft, life rafts and military equipment for example rockets. The dye commonly tried is fluorescein, which is a water soluble synthetic dye. Different compositions of the dye for better efficiency and longer duration of fluorescence in diluted form are under trials (Swinton; Robert J, US patent No.

5405,416 published on April 11,1995 and International patent number W09010044 disclosed on 7 July 1990). Hyosu et al. (April 5,1977 US patent 4,016, 133) has prepared fluorescent coloured resin particles.

Another use of marine dyes as undersea probes is reported by Crosby, David A and Ekstrom, Philip A, in US patent No. 5321268 published on 14 June 1994. The probe device includes a central optical fibre containing a fluorescent dye enclosed in a transparent or translucent, protective and fouling resistant sheath. This can be attached to marine animal for collecting data as to light intensity and temperature in regions where the marine animals travel.

Some authors have used UVA in photo-chemotherapy for skin cancers. Kowalzick L ; Ott A; Waldmann T; Suckow M; Ponnighaus J, M. Vogtlandklinikum Plauen, disclose Puna-bath photo-chemotherapy in lymphomatoid papulosis (a skin cancer) where UVA treatment has shown improvement (Elsevier Science B. V, 2000). UV A sun beds are widely used by patients with psoriasis.

Sabatelli, Anthony D. (US patent No. 5,210, 275 pub. date May 11,1993) disclosed a chromatophore sunscreen composition for preventing sunburns. The chromatophore had the ability to absorb UVA and UVB wavelength radiations.

Fluorescent dyes are very useful in labelling of molecular probes for fluorescence microscopy. Fluorescence microscopy also known as Reflected light fluorescence or epifluorescence microscopy which is of great value for non-radioactive in situ hybridisation, because of its high sensitivity and ability to excite three different immunofluorophore with spectrally separated emissions. It makes the multiple detections possible (Chapter II. Non-radioactive In Situ Hybridisation Application Manual. Boehringer Mannheim GmbH, Biochemica, printed in Germany, 1992). The principle behind is that when the specimen is irradiated by the excitation wavelength corresponding

to Stoke's Law, which explains that the wavelength of the fluorescent radiation is always longer than that of the excitation radiation ("Fluorescence"by George L. Clark, 1966 in Encyclopaedia of chemistry, 2nd ed. Page 435-436). Chapter V in: In Situ Hybridisation Application Manual. Boehringer Mannheim GmbH, Biochemica, printed in Germany, 1992, Page 23-62 and Olympus Optical Co. Ltd, Tokyo Japan. Catalogue."Instructions BX-FLA Reflected Light Fluorescence attachment"Page 16. 1999 described variety of non-radioactive fluorochrome stains in use these days.

Different stains are used for different excitation cubes of the fluorescent microscope. For example DAPI (DNA staining, emits blue colour), Fluorescein-dUTP ; Hoechest 33258,33342 are seen under excitation with 330-385 excitation cubes; FITC, Acridine Orange (for DNA, RNA emits greenish/yellowish hues), Auramine under 450480 excitation cube and Rhodamine, TRITC and Propidium iodide (DNA, emits orange hues) under 510-550 excitation cube.

Rosenblum, Barnett B and Spurgeon Sandra L; Lee Linda G; Benson Scott C; Graham Ronald J used a set of 4, 7-dichlororhodamine compounds useful as fluorescent dyes as molecular probes in international patent number W00058406 published on 5th October, 2000.

LaClair; James J. (US Patent No. 6,140, 041 published on October 31,2000 and W09938919) disclosed synthesis of a fluorescent dye and its application in protein labelling, DNA labelling, single molecule spectroscopy and fluorescence.

The applicants have adopted a different approach; the dye reported in the present invention is a natural dye and not synthetic. It is from a marine animal and not from a plant or microbes. The partially pure dye is extracted from the cells of the skin of the invertebrate directly. This is a first report for a natural dye from a marine animal which is a fluorescent dye. The marine animal source a holothurian, sea cucumber called Holothuria scabra is a new source. Unlike most other fluorescent synthetic dyes known, our dye does not need to be mixed with another dye for getting different fluorescence hues at different wavelengths. It emits three different coloured fluorescences at three different excitation wavelengths, which can have multiple uses. Further even amongst the naturally known fluorescent dyes like the most popular green fluorescent protein (GFP) from a jelly fish, our dye is non proteinaceous in nature and is more stable at the room temperature for months and does not get contaminated by microbes. It has also qualities of a bio-surfactant.

Another important feature of the dye is that after excitation in the lower UV spectral ranges of wavelengths (UVB) it emits fluorescence in the UVA wavelength range. Both of these absorption and emission ranges can be put to selective applications depending upon which UV a spectrum is preferable in a particular situation.

One important aspect of the dye is its making compositions and kits for non radioactive labelling of molecular probes and counterstaining. At different wavelength excitations it gives the effect equivalent to colour of DAPI, FITC and PI. The dye is three in-one though it is a single dye. Actually, this single dye covers the colours of wavelength spectrum of 123-flurochromes presently known in the market (see Bitplane products (Fluorochrome) on the Internet http://www. biplane. ch/public/support/standard/Fluorochrome. htm.

Yet another aspect is its use as a fluorochrome stain in epifluorescence microscopy, which is reported here for the first time for any marine natural dye. This application provides a simple and quick method of checking cytogenetical preparations for multiple uses like molecular diagnostics using fluorescent in situ hybridisation techniques, rapid diagnosis of bio-contamination in tissue cultures, industrial preparations, water quality check in laboratory and wild conditions.

Yet another aspect of the dye is its use as a component of the non-radioactive labelling kits for advanced molecular biology applications.

The main object of the present invention is to provide a novel fluorescent dye obtained from the sea cucumber Holothuria scabra.

Another object of the invention is to provide a process for extraction, partial purification and characterization of the said natural dye/pigment from the marine animal Holothuria scabra.

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

Yet another object of invention is to see its insecticidal and pesticidal effects.

Yet another object of the invention is its application for veterinary remedies.

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

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

Yet another object is to observe the three different fluorescence coloured emissions of the dye in UV and visible ranges of epifluorescence microscopy cubes.

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.

Yet another object of the invention is bio-surfactant nature analysis.

Still another object is to develop kits containing the fluorescent dye as non radioactive label for molecular probes.

Accordingly, the invention provides a novel fluorescent dye obtained from the sea cucumber Holothurie sabra. The invention also provides a process for extraction, isolation and characterization of the said dye. Further, the invention provides compositions containing the said dye.

After much research, the applicants have now identified a novel fluorescent dye obtained from marine animals, especially from invertebrates and more specifically from the sea cucumber Holothuria scabra.

Subkingdom: Metazoa Phylum: Echinodermata Sub-Phylum: Eleutherozoa Class: Holothuroidea Subclass: Aspidochirotacea Order: Aspidochirota Amongst these orders sea cucumber Holothuria scabra belongs to: Order: Aspidochirota Family: Holothuriidae Genus: Holothuria Species: scabra The invention further provides a novel fluorescent dye which is obtained from the skin of the animal. It also describes the physical and chemical nature of the dye and its stability in direct light, high and low temperature. The said dye has three coloured 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 contamination and cytogenetical screening. 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, epifluorescence microscopy for single and double staining of chromosomes, cells and tissues, fluorescence in situ hybridisation applications, bio-surfactant, bio-contamination and leakage check, photo chemotherapy, novel remote sensing devices, underwater probes, life saving devices, mark the location of crashed aircraft, life rafts and military equipment for example rockets, various fluorescence applications in sub zero temperature conditions and many more.

The invention describes fluorescent dye obtained from marine animals that either absorb sunlight for their physiological functions or are exposed to longer durations 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.

The invertebrate animals who do not have an extra outer armour 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.

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, epifluorescence microscopy, photochemotherapeutics, component of new instrumentation devices for land and underwater probes, cosmetic industry, food industries and armed forces etc The said marine invertebrate is an echinoderm taxonomically called Holothuria scabra belonging to the class Holothuroidea. The product of the invention is a novel 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 said. 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 kind from marine organisms.

As used herein the term dye is used for a pigment which does not get decolourised by a reducing agent. The said dye imparts colour to the fibre, cellulose etc. It is called a natural dye as the source is from a marine animal found commonly in the nature along shores of the world and is not a synthetic pigment. A fluorescent dye is one that 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.

Multiple coloured fluorescence, as used herein, means the emission of different coloured light when excited at different ranges of wavelengths. It emits blue, yellow and orange coloured hues of fluorescence at excitations with different spectra of UV and visible light. Bio-surfactant means a dye solution which if shaken provides foam like soap and shows anti-microbial quality. The molecular diagnostics as used herein means the use of the dye as a non-radioactive label of molecular probes for fluorescent in situ hybridisation applications in molecular cytogenetics and as markers in microarrays, and molecular biological studies. The epifluorescent microscopy here pertains to the microscopic studies of cytogenetical preparations of slides by using the present dye as a stain and recording different coloured fluorescence when observed under different cube configurations emits a particular coloured emission on excitation with known fluorochrome. The fluorochrome cubes WUB, WB, WG are the designated filter cube configurations of the Olympus BX-FLA reflected light fluorescence attachment for different wavelengths.

Accordingly, the invention provides a method for extraction, purification and characterization of a natural fluorescent dye, which comprises: (i) collection of the material from field and maintenance in the laboratory conditions, (ii) extraction of the pigment from the skin of the echinoderm sea cucumber Holothuria scabra, and (iii) partial purification of the dye.

The bioactive extract of the invention is obtained from the marine sea cucumber Holothuria scabra. This extract is useful as a natural fluorescent dye and has the following characteristics : 1. decolourisation by a reducing agent,

ii. not a synthetic compound, iii. crude extract of the dye is yellowish green in colour, iv. partially purified dye being reddish brown coloured powder when seen with the naked eye in the daylight, v. under tube light some hues of green are emitted, vi. amorphous in nature, vii. soluble in water, viii. insoluble in the organic solvents like ethanol, methanol and acetone, ix. is negatively charged, x. has a pH of 6.5, xi. presence of a phenolic group, xii. absence of a quinonoid ring, xiii. absence of aromatic amine groups, xiv. non-proteinaceous in nature, xv. reducing sugar is absent, xvi. dye has nature of a biosurfactant, xvii. dye also showed antimicrobial qualities and when antimicrobial assay was performed, showed zone of inhibition, xviii. pigment cum dye is a fluorescent dye and emits fluorescence when excited with different wavelengths of UV and visible spectral ranges on a spectrophotometer, xix. UV, visible spectroscopy from 300nm-700nm and the peaks are marked at 379 nm and 439nm wavelengths, xx. UV, visible spectroscopy from 250 nm-350 nm and the peaks are at 272 nm and 299 nm wavelengths, xxi. fluorescent spectroscopy in the UV and visible spectra, when excited with UV 270 nm wavelength the fluorescence is emitted in the 324nm-380nm range which comes under the UVA wavelength range of ultraviolet rays of the sunlight, xxii. with excitation wavelength 450nm in Fluorescent spectroscopy the fluorescence emission occurred at 500nm-580 nm with maximum intensity,

xxiii. with excitation wavelength 540nm in Fluorescent spectroscopy, the fluorescence emission occurred at 500nm-620 nm with maximum intensity, xxiv. with excitation wavelength 555 nm in Fluorescent spectroscopy, the fluorescence emission occurred at 575 nm-620 nm with maximum intensity, xxv. physical checking of Whatman Filter no. 1 dipped with dye concentration 1: 40000 dilution under UV transilluminator and Gel Documentation system with UV bulbs of 260nm-280nm range emit bluish green hue colour of fluorescence, xxvi. emits three different coloured fluorescence at 3 different wavelengths of the UV and visible ranges of the fluorescent cubes of an epifluorescence microscope, xxvii. fluorescence blue colour emission occur in the 380nm-400 nm range of UV A

when excited under ultra violet cube WU-330 nm-385 nm excitation range, xxviii. fluorescence yellow colour emission occurs in the 500nm-570nm range when excited under WB cube of450nm-480 nm excitation range, xxix. fluorescence orange colour emission occurs in the 570 nm-650nm range when excited under WG cube of510nm-550 nm excitation range, xxx. the dye emits hues of greys under the ordinary transmitted light bulb of the epifluorescence microscope when seen under 10X objective, xxxi. the dye emitted these fluorescence colours even at a dilution range of 1: 40000 times (i. e. Igm powder of dye dissolved in 40 litres of ultrapure water), xxxii. the fluorescence of the extract persisted even after at least lyear at the room temperature, xxxiii. the fluorescence of the dye is highly photostable and does not get deteriorated by long exposures to direct light, and xxxiv. the fluorescence of the dye does not change even when frozen at 20OC, a temperature at which the molecules are unable to attain the energy necessary for activation like in extracts from luminescent organisms.

The physical and other characteristics of the dye may be assessed by the following steps: (i) Structural analysis of the dye,

(ii) Biosurfactant analysis, (iii) Antimicrobial test, (iv) Visible spectroscopy of the dye, (v) Fluorescence spectroscopy of the dye, (vi) Physical checking of emission under a UV transilluminator 260-280 nm range, (vii) Preparation of the cytogenetic slides by air dried method, (viii) Staining of slides with the dye, (ix) Epifluorescent microscopic screening of the cytogenetic slides under fluorochrome cubes WU, WB, WG and Bright field, (x) Microphotography of emitted fluorescence in the areas of slides without any cytogenetic material, (xi) Microphotography of emitted fluorescence of the cytogenetic slides under fluorochrome cubes WU, WB, WG and Bright field, and (xii) Checking of wavelength ranges of the fluorescent hues of emission and wavelength ranges of the excitation ranges of fluorochrome cubes.

Thus the invention provides a natural fluorescent dye of marine animal origin, which emits three different coloured fluorescence in the hues of blue, yellow and orange when excited with three different ranges of wavelengths in the UV and visible light spectral cubes of an epifluorescence microscope. The invention further relates to the peaks of emission at the nearly same ranges of excitation wavelengths by recording readings of a fluorescence spectrophotometer and the visible light spectrophotometer respectively. The invention further relates to the epifluorescence microscopy of cytogenetic material on airdried preparations by using this dye as the epifluorescent microscopic stain. This dye could be used in making non-radioactive labelling kits for molecular diagnostics by fluorescent in situ hybridisation in various molecular, biomedical and engineering sciences.

In an embodiment the source of the dye is an invertebrate marine animal belonging to Subkingdom : Metazoa, Phylum Echinodermata ; subphylum Eleutherozoa, Class

Holothurodea. Name : Holothuria scabra.

In yet another embodiment the Holothuria scabra is selected from the group compnsing 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.

In yet another embodiment skin of Holothuria scabra is separated and weighed. To 15g of skin by wet weight 250 ml of 50% alcohol is added and filtered under vacuum by using a peristaltic pump working at the rate of 200 rpm.

In yet another embodiment the extract is evaporated to one third of its volume keeping on a water bath at 80 degree centigrade i. e. from 250 ml concentrated to 80 ml. It takes about 3 hours of duration for evaporation.

In yet another embodiment 100 ml ethanol 99.5% is added to 80 ml of the concentrate of the extract and allowed to precipitate overnight.

In yet another embodiment the concentrate with the precipitate is centrifuge at 1500 rpm for 4-5 minutes and the top layer is decanted. The precipitate is evaporated to the dryness on water bath at 80 degree Centigrade for 5 minutes 250 ml of 50% ethanol extract provide 2.5g of dye on evaporation.

In yet another embodiment the partially pure dye is scooped out with the help of a spatula and stored in a dry glass vial at the room temperature.

In another aspect the physical nature of the dye is recorded. The pure dried dye is reddish brown in colour in the day light. Under tube light a hue of green is observed. The dye is soluble in water, and insoluble in organic solvents like pure ethanol, methanol, chloroform and acetone. It is amorphous in nature. It has pH of 6.5 in aqueous solution.

In yet another aspect structural analysis was done by chemical method. The dye is dissolved in distilled water in a concentration of 2mg/ml and checked for chemical nature.

In yet another embodiment neutral ferric chloride was added and purple colouration was observed. It proved that phenolic group is present.

In another embodiment ?-mercaptoethanol (Beta mercaptoethanol) reducing agent was added. No decolouration of the compound occurred. This proved that quinonoid ring is absent and the pigment is a dye.

In another embodiment the diazotization was done by adding 0. 1 N HCI and NaNOz (Sodium nitrite) and alkaline solution of beta (p) naphthol was added to it. No precipitation was observed. This proved absence of the amine group.

In another embodiment the concentrated dye solution in a concentration of 10 mg/ml was heated and no precipitation or coagulation was observed. This proved that the compound is

non-proteinaceous in nature. To the same solution add a drop of concentrated HCL and added Fehling's solution. No colour change proved that the reducing sugar is absent.

In another embodiment the biosurfactant nature of the dye was observed by its making a foam while added to water and shaken.

In another embodiment the anti-microbial disc test was performed and the zone of inhibition was observed.

The applicants studied the nature of the dye and found that it gave multicoloured emissions at different wavelengths of excitations, which are comparable to the fluorochrome microscopic stains already in the market. The blue coloured fluorescence of the present dye is comparable to the emission of same colour by DAPI fluorochrome at the same wavelength excitation, used as components of the non-radioactive labelling kits of biochemistry, cell biology, immunochemistry, and molecular biology. The yellowcoloured fluorescence of the said dye in the visible range is comparable to the same coloured emissions of auramine used as components of the non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology. The yellow-coloured fluorescence of the said dye in the visible range is comparable to the same coloured emissions of FITC used as components of the non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology. The orange coloured fluorescent emission is comparable to the orange fluorescence colour of Propidium Iodide fluorochrome used as components of the non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology. A dye as claimed in claim 1 wherein the orange-coloured fluorescent emission is comparable to the orange fluorescence colour of TRITC fluorochrome, used as components of the nonradioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology. 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 including DAPI, Hoechest 33258, Hoechest 33342, FITC, acridine orange, auramins, Rhodamine, TRITC, and propidium iodide, which are now in the market. The dye, under ordinary light of microscope the hues of greys produce a phase contrast effect which is useful in rapid screening of cytogenetical, cytological, and histochemical slides and save expenses on the extra phase contrast accessory component of microscope The

fluorescence colour emissions follow Stoke's law of fluorescence. The microphotographs with Kodak film rolls shows hues of the adjacent colour emission wavelengths such as blue colour fluorescence under the epifluorescence microscope in microphotograph the hues of green. The microphotographs with Kodak film rolls show hues of the adjacent colour emission wavelengths like when seen yellow colour fluorescence under the epifluorescence microscope in microphotograph the hues of green also come. The orange fluorescence colour under the epifluorescence microscope in microphotograph the hues of red also come. The cytogenetic slides seen for fluorescence gives a counterstain effect of cells with the background where no specimen but only dye is present.

In another aspect, UV visible spectroscopy with a wavelength from 300-700nm was performed (Fig. 3). The peaks are marked at 379 nm and 439nm wavelengths.

In another embodiment the UV spectroscopy was performed with a wavelength from 250-350nm (Fig. 4). The peaks are marked at 272 nm and 299 nm wavelengths In yet another aspect, the fluorescence spectroscopy was performed with an excitation wavelength 270nm. The fluorescence occurred at 324 nm-380 nm with maximum intensity (Fig. 5).

In another embodiment, the fluorescence spectroscopy was performed with an excitation wavelength of 450nm. The fluorescence occurred at 500nm-580nm with maximum intensity (Fig. 6).

In another embodiment, the fluorescence spectroscopy was performed with an excitation wavelength of 540nm. The fluorescence occurred at 500 nm-620 nm with maximum intensity (Fig. 7).

In another embodiment the fluorescence spectroscopy was performed with an excitation wavelength of 555 nm. The fluorescence occurred at 575 nm-620 nm with maximum intensity (Fig. 8).

In another embodiment a Whatman number 1 filter paper is soaked in the extract of the dye and observed under the UV transilluminator with UV bulbs of 260nm-280nm wavelength range. It emitted blue fluorescence.

In yet another aspect the epifluorescence microscopic studies are made by using this dye as a stain in the dilutions of 1: 10000 (1 g per 10 litre) and recording emissions of light when excited by different cubes and compared the colour hues with the known fluorochromes.

Different stains are used for different excitation cubes of the fluorescent microscope. For example DAPI (DNA staining, emits blue colour), 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 nu -550 nu excitation cube.

In an embodiment of this invention, epifluorescence microscopic screening of the cytogenetic slides is done by putting a drop of the diluted extract and excitation with the WU filter having spectral range of 330-385 nm wavelengths.

In another embodiment, epifluorescence microscopic screening of the cytogenetic slides is done by putting on a drop of the extract and then allowing excitation with the WB filter having spectral range of 450-480 nm wavelengths.

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

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

In yet another embodiment epifluorescence microscopic screening of the cytogenetic slides stained with the dye is done by observing hues of the fluorescence colour emitted by the respective excitations.

In another embodiment the excitation with the WU 330 nm-385 nm range emitted fluorescence is in the 380 nm-400 nm range.

In another embodiment the excitation with the WB filter having spectral range of 450 nm-480 nm emitted fluorescence in the 550 nm-570 nm range.

In yet another embodiment the excitation with the WG filter having spectral range of 510 nm-550 nm emitted fluorescence in the 600 nm-650 nm range.

In another embodiment epifluorescent microscopic screening of the cytogenetic 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.

In yet another aspect of the invention microphotography of emitted fluorescence in the areas of slides without cells under WU 330 nm-385 nm range is done by Kodak film of 400 ASA speed with an exposure varying from 50 seconds to 60 seconds. The hues of blue fluorescence were emitted.

In another embodiment microphotography of emitted fluorescence in the areas of slides without cells under WB 450 nm -480 nm range is done by Kodak film of 400 ASA speed with an exposure varying from 50 seconds to 60 seconds. The hue of yellow fluorescence was emitted.

In another embodiment microphotography of emitted fluorescence in the areas of slides without cells under WG 510 nm -550 nm range is done by Kodak film of 400 ASA speed with an exposure varying from 50 seconds to 60 seconds. The hue of orange fluorescence was emitted.

In another embodiment microphotography of emitted fluorescence in the areas of slides without cells under Bright field hues of grey was emitted.

In another embodiment microphotography of emitted fluorescence in the areas of slides with cells under WU 330 nm-385 nm range is done by Kodak film of 400 ASA speed with an exposure varying from 50 seconds to 60 seconds. The hue of blue fluorescence was emitted.

In another embodiment microphotography of emitted fluorescence in the areas of slides with cells under WB 450 nm-480 nm range is done by Kodak film of 400 ASA speed with an exposure varying from 50 seconds to 60 seconds. The hue of yellow fluorescence was emitted.

In another embodiment microphotography of emitted fluorescence in the areas of slides with cells under WG 510 nm-550 nm range is done by Kodak film of 400 ASA speed with an exposure varying from 50 seconds to 60 seconds. The hue of orange fluorescence was emitted.

In another embodiment microphotography of emitted fluorescence in the areas of slides with cells under Bright field hue of grey was emitted.

In yet another aspect of the invention the 1: 10000 times dilutions of the dye when prepared in distilled water and used as a stain the coloured fluorescent emissions occurred in the UV and visible ranges of the epifluorescence microscope.

In still another embodiment, the dye is diluted with water in the ratio 1 : 40, 000 times and this gives fluorescence of three colours at three different wavelengths.

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: 40000 in ultrapure water to obtain fluorescence of three colours at three different wavelengths and a phase contrast effect under transmitted light.

The invention also provides a composition comprising a bioactive extract obtained from the marine sea cucumber Holothuria scabra together with conventional additives and useful for the preparation of flexible polyvinyl chloride film that exhibits fluorescent colours.

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.

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.

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.

In still 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 photo chemotherapy of skin cancers.

In yet another embodiment the invention provides a cosmetic composition comprising a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives.

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 hybridisation kits for molecular diagnosis.

In yet another embodiment the invention provides a composition comprising a

bioactive extract obtained from the marine sea-cucumber llolothuria scabra together with

conventional additives and useful as a component of non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry and molecular biology.

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

In yet another embodiment the invention provides a composition comprising a bioactive extract obtained from the marine sea-cucumber Holothurie sabra together with conventional additives and useful as a counterstain of DIG-labelled oliogonucleotide probes and anti-DIG Fab-fragments.

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

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

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.

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.

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 rapid check ofbiopollutants under field conditions.

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 competitive inhibitor of cholinesterases.

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 antimicrobial compositions.

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 biosurfactant in toiletry compositions.

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 natural colorant.

In yet another aspect of the invention the 1: 10000 times dilutions of the dye when prepared in distilled water and used as a stain the coloured fluorescent emissions occurred in the UV and visible ranges of the epifluorescence microscope.

In still another embodiment, the dye is diluted with water in the ratio 1: 40,000 times and this gives fluorescence of three colours at three different wavelengths.

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: 40000 in ultrapure water to obtain fluorescence of three colours at three different wavelengths and a phase contrast effect under transmitted light.

The invention also provides a composition, comprising: a bioactive extract obtained from the marine sea-cucumber Holothuria scabra together with conventional additives and useful for the preparation of flexible polyvinyl chloride film that exhibits fluorescent colours.

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.

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.

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

In still 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 photo chemotherapy of skin cancers.

In yet another embodiment the invention provides a cosmetic composition, comprising: a bioactive extract obtained from the marine sea cucumber Holothurie sabra together with conventional additives.

In yet another embodiment the invention provides a composition, comprising : a bioactive extract obtained from the marine sea cucumber Holothuria sabra together with conventional additives and useful as a fluorescent probe in situ hybridisation kits for molecular diagnosis.

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 labelling and detection kits of biochemistry, cell biology, immunochemistry and molecular biology.

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 immune fluorescent detections.

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-labelled oliogonucleotide probes and anti-DIG Fab-fragments.

In yet another embodiment the invention provides a composition, comprising: a bioactive extract obtained from the marine sea cucumber Holothuria sabra together with conventional additives and useful in single and multiple cell quantitative fluorescence in flow cytometry.

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.

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 ocontamination in the health food mdustry, cosmetic industry, pharmaceutical and chemical industries.

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.

In yet another embodiment the invention provides a composition, comprising: a bioactive extract obtained from the marine sea cucumber Holothuria sabra together with conventional additives and useful for a rapid check ofbiopollutants under field conditions.

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 competitive inhibitor of cholinesterases.

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 antimicrobial compositions.

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 biosurfactant in toiletry compositions.

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 natural colorant.

The invention is further described with reference to the following tables: Table 1. (a) & (b) Structural analysis of the dye by chemical method for presence or absence of Quinonoid ring, phenolic and amine group.

Table 2. Structural analysis of the dye by chemical method for checking absence of Quinonoid ring, proteinaceous/non-proteinaceous nature and presence/ absence of the reducing sugar.

Table 3. The colour of the different coloured fluorescence of the dye used as stain when excited with different wavelength cubes of the Olympus epifluorescence microscope.

The invention is further illustrated with reference to the following figures : Figure 1 : Sea cucumber Holothuria scabra fresh from the field before Extraction of the dye.

Figure 2: Sea cucumber Holothurie scabra after 4 time extraction of the dye.

Figure 3 : Scanning in UV visible region Figure 4: Scanning in the UV region Figure 5 Fluorescence spectrophotometry. Excitation wavelength 270 nm.

Figure 6: Fluorescence spectrophotometry. Excitation wavelength 450 nm.

Figure 7: Fluorescence spectrophotometry. Excitation wavelength 540 nm.

Figure 8: Fluorescence spectrophotometry. Excitation wavelength 270 nm.

Figure 9: Whatman filter paper 1 is cut round, dipped at the tip in the dilute crude extract of the dye and seen under UV radiation in a Gel Doc. The arrow shows the fluorescence, photographed through a UV filter. The lower part is the control without the dye.

Figure 10: Whatman filter papers, used for filtration of the extract and seen under UV transilluminator (260-280rim wavelength range bulbs). The arrows show fluorescence. The lower filter paper is without any extract kept as control.

Figures 11-14 : Epifluorescence microscopic blue fluorescent emissions with WU cube having excitation range of 330 nm-385 nm. Fig. 11 is fluorescence of dye without any specimens. Fig 12 is with cells seen under 10X objective. Fig.

13: the same seen under 40 X objective and Fig. 14 cells seen under 100 X oil immersion objective.

Figures 15-18: Epifluorescence microscopic greenish yellow fluorescent emissions with WB cube having excitation range of 450 nm-480 nm. Fig. 15 is fluorescence of dye without any specimens. Fig 16 is with cells seen under 40X objective. Fig. 17 & 18: the same as seen under 100 X oil immersion objective.

Figures 19-22: Epifluorescence microscopic hues of orange fluorescent emissions with WG cube having excitation range of 510-550nm. Fig. 19 is fluorescence of dye without any specimens. Fig 20 is fluorescence with cells seen under I OX objective. Fig. 21: the same seen under 40 X objective and Fig. 22. cells seen under 100 X oil immersion objective.

Figure 23 : Cells as seen through bright field under 10X objective. The hue of grey-a phase contrast effect was seen.

Figure 24 : The same cells as seen through bright field under 100X oil immersion objective.

This invention pertains to the process of extraction, purification and characterization of a new pigment, which is a natural dye from an echinoderm (Holothuroidea: Holothuria scabra) widely distributed along the central west coast of India and the Indo-pacific regions of the world.

The invention further provides a novel fluorescent pure dye from the skin pigment of the animal which can be repeatedly extracted 3-4 times from the same specimen by storing below-20 degree Centigrade, thus saving over exploitation of natural resources.

The present invention also contemplates that the said dye has three coloured fluorescent emissions at three different excitation wavelengths of UV and visible light spectra equivalent to emissions by three different fluorochromes (DAPI, FITC and PI) currently sold in the market for fluorescent microscopy.

Thus the dye can be commercialised as three in one fluorochrome dye of epifluorescence microscopy for single and double staining of chromosomes, cells and tissues following simple protocols.

The present invention also contemplates the use of the dye in non-radioactive labelling of protein, DNA and RNA probes for fluorescent in situ hybridisation applications in molecular biology. Thus in a preferred mode of use the dye can be a component of molecular labelling and detection kits, most of which are imported and sold at high rates. These labelling kits are widely sought after for molecular diagnostics using rapid molecular cytogenetic and microarrays techniques.

In yet another preferred mode of utility, dye can be advantageous in making compositions of cosmetics for absorption of UVB from the sunlight Yet another advantage of the dye is that its fluorescence is visible even in very dilute solutions (1: 40000). This property can be utilized in life saving devices as a component of life jackets and to mark the location of crashed aircraft, life rafts and military equipment for example rockets, leaking checks in the industries. The invention would be useful for quantitative measure of fluorescence In flow cytometer for single and multiple

cells. The invention would be also advantageous in quick estimations ofbiocontamination in natural and controlled environments like tissue cultures, pollution, and industrial contamination in health, food and cosmetic industries. The ability of the dye to emit fluorescence in the UVA range when excited with lower wavelengths of UV irradiation is useful for selective photo chemotherapy of skin cancers.

In another preferred mode the dye can be of use as a component of sunscreen compositions in cosmetic industry.

Yet another utility is that the dye is a biosurfactant and can be used in antimicrobial toiletry and compositions.

In another preferred mode of use the dye has a long shelf life at the room temperature as checked by fluorescent spectrophotometric analysis.

In a yet another preferred mode of use the fluorochromes present will give natural colour to the cosmetics and save expenses on colour additives.

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

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: The methods of extraction, partial purification, characterization of the dye and the details of the experiments performed to check fluorescent effects of the dye by spectroscopic analysis and epifluorescence microscopy are disclosed: Example 1 Collection of the material.

Subkingdom: Metazoa Phylum: Echinodermata Sub-Phylum: Eleutherozoa Class: Holothuroidea Subclass: Dendrochirotacea Order: Dendrochirotida Genus: Holothuria Species: sabra

The animals belonging to the were collected from the shores of central west coast of India during a low tide. These were brought to the laboratory and maintained in glass tanks containing seawater of salinity 30-32 per par (30%) till taxonomic identification and further use. The animals were adult and sexually mature.

Example 2 Extraction of the pigment Two methods were tried.

1) In our initial experiments the animals after collection were frozen at-20 degree Centigrade and when thawed partial pigment came out in the tray. Which was processed and this way from one animal 3-4 times pigment could be removed. Figs.

1 & 2 show the fresh and 4 times used animal for extractions.

2) In the second method the animals were first washed with tap water and then with Milliq water (ultrapure water). The body was cut open with the sharp scissors and body wall was separated from the other viscera. From body wall the with the help of sharp razors epidermal skin portion was peeled off and stored in-20 degree Centigrade refrigerator if not processing further immediately. it was then placed in a beaker and

50% vol/vol ethanol alcohol and MQ water mixture was added to it in the following ratios.

15gm animal skin : 250 ml of 50% ethanol alcohol.

Example 3 Filtration of the pigment solution This step was performed to remove the debris of cells and some of the suspended and precipitated impurities. Centrifuge it down so as to make a clear solution and precipitate down all suspended things.

The coloured solution was decanted and filtered with a micro filtration unit (Vensil make) glass filter with the help of a peristaltic pump. The filtrate was placed on an orbital shaker

and left for half an hour at a rotation of 200rpm.

Example 4 Concentration of the pigment The coloured solution was then placed on a water bath at 80 degree Centigrade and concentrated to one third of it's volume. This also evaporated the alcohol present. The concentrate was again subjected to filtration with the same filtering apparatus.

Example 5 Purification of the dye The solution of pigment will contain impurities like NaCl, MgCl2, MgS04 and other watersoluble compounds. It was seen that polar organic solvents like alcohol (dehydrated), acetone if added to the concentrated solution of pigment it will rapidly be precipitated. Then it can be separated from the seawater salts. This is to purify the pigment for spectroscopic analysis.

The concentrated solution prepared by example 5 described above was taken in a separating funnel of 500ml and to this was added ethanol (80ml of concentrated supernatant + 100ml of 99.5% ethanol). Tilted the separating funnel to gently mix the contents and collected the precipitate overnight. The concentrate with the precipitate is

centrifuge at 1500rpm for 4-5 minutes and the top layer is decanted. The precipitate was again dissolved in 5ml of MQ water and again 100% ethanol was added until the precipitation is complete. This was again centrifuged and the precipitate was collected.

This step is repeated 3-4 times to purify the pigment. The precipitate is evaporated to the dryness on water bath at 80 degree Centigrade for 5 minutes. The pure dye is scooped out with the help of a spatula and stored in a dry glass vial at the room temperature. 250ml of 50% ethanol crude extract from example 2 provided 2.5g of partially purified dye on evaporation in a powder form.

Example 6 Physical characteristic of the compound The crude extract is yellowish green in colour. The physical nature of the pure dried dye when recorded with the naked eye is reddish brown in colour in the day light. Under tube light a hue of green is observed. The dye is soluble in water and insoluble in organic

solvents like pure ethanol, methanol and acetone. It is amorphous in nature. It has pH of 6.5 and has a negative charge.

Example 7 Structural analysis of the dye by chemical methods Experiment 1: CHNS elemental analysis of the dye was performed and results are given in Table 1 (a) and (b).

Experiment 2: The dye is dissolved in MQ water at a concentration of 2mg/ml and checked for chemical nature. The presence and absence of certain groups was tested and results are given in Table 2. Whenever a test showed negativity, the higher concentration of dye was used and experiment performed again.

For example to 2mg/ml solution of dye Beta-mercaptoethanol (reducing agent) was added. No decolouration of the compound occurred. This proved that Quinonoid ring is absent and the pigment is a dye.

In experiment 3, the concentrated dye solution at a concentration of 10 mg/ml was heated and no precipitation or coagulation was observed. This proved that the compound is nonproteinaceous in nature.

In experiment 4 to the same solution a drop of concentrated HCI and Fehling's solution was added. No colour change proved that the reducing sugar is absent.

Example 8 Checking electric charge of the Dye The charge of the compound was analysed by gel electrophoresis. Dye samples (10mil) are loaded in 1% agarose gel prepared with 0.5 X TBE. The gel was allowed to run for an hour at 65 volts. It was removed from the gel casting system and observed by eye as well as under a UV transilluminator. It was found that the dye was moving towards positively charged electrode, so the dye itself is a negatively charged compound. Hence it was getting attracted towards positively charged electrode.

Example 9 Biosurfactant analysis The biosurfactant nature of the dye was observed by its making a foam while added to water and shaken. The solution gave feeling of soapiness.

Example 10 Antimicrobial test As the marine dye is a phenolic compound and phenolic compounds are generally having antimicrobial activity antimicrobial assay was performed with this compound and the zone of inhibition was observed.

E. coli (wild type) culture was grown overnight in MacConkey's broth 50ml in a conical flask (100 ml). Antibiotic assay agar medium was prepared and sterilized. It was then brought to a temperature of 50 degree centigrade and 1ml ofE. coli (wild type) culture was added to it. The culture was mixed with antibiotic assay agar medium and was allowed to

solidify. 1 Omglml of the sample was prepared and soaked in filter paper disks. It was then placed upon the said antibiotic assay agar medium seeded with E. coli. It was then incubated at 37 degree centigrade in an incubator for 24 hours. Zone of inhibition surrounding the filter disk was observed. This proved that the said dye was having antimicrobial activity against Gram Negative organisms like E. coli.

Example 11 UV Nisible spectroscopy of the dye Instrument used: Genesys2 UV spectrophotometer. 2mg/10 ml solution was prepared in volumetric flask and spec readings was taken in UV visible range by using a quartz cuvette and adding 2ml of the solution. The control was ultra pure water. UV, visible spectroscopy from 300nm-700nm wavelength (Fig. 3 & 4) was performed. The peaks are marked at 379 nm and 439nm wavelengths. UV visible spectroscopy was performed from wavelength 250-350 nm (Fig. 3 & 4). The peaks are marked at 272 nm and 299 nm wavelengths.

Example 12 Fluorescence spectroscopy of the dye Apparatus used: Hitachi Fluorescence spectrophotometer. The fluorescence spectroscopy was performed at different wavelength ranges of the visible and UV spectra and emission ranges noted. It was found that with excitation wavelength of 270nm. The fluorescence occurred at 324-380nm with maximum intensity (Fig. 5).

In the fluorescence spectroscopy, with excitation wavelength 450nu the fluorescence occurred at 500-580 with maximum intensity (Fig. 6).

In the fluorescence spectroscopy, with excitation wavelength 540nu the fluorescence occurred at 500-620 nm with maximum intensity (Fig. 7) and with the fluorescence spectroscopy at excitation wavelength 555 nm the fluorescence occurred at 575-620 nm with maximum intensity (Fig. 8).

Example 13 Physical checking of emission under a UV transilluminator and Gel Documentation system A Whatman no. 1 filter paper was cut and dipped in diluted crude extract and seen under a gel doc UV light. It was clearly seen that as the dye progressed seeping in the fluorescence area progressed further (Fig. 9).

In another test the filter papers used for filtration were seen under the UV transilluminator with 260-280 nm UV range bulbs. The bluish green hued fluorescence was noticed. (Fig. 10).

Example 14 Epifluorescence microscopy The epifluorescence microscopic studies are made by using this dye as a stain in the dilutions of 1 : 40000 and recording emissions of light when excited by different cubes and compared the colour hues with the known fluorochromes. The cytogenetical an-dried preparations of a fixed tissue were made. To this was added a drop of the stain and placed a covershp. The screening was done using excitations of UV light and visible light spectra by WU, WB and WG cubes of the Olympus reflected hght.

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

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

WG cube's wavelength range was 5 lOnn-550 nm.

Example 15 The emission ranges at different excitation ranges were calculated It was seen that: Excitation with the WU 330 nm -385 nm range emitted fluorescence in the 380-400 nm range.

Excitation with the WB filter having spectral range of 450-480nm emitted fluorescence in the 500-570nm range.

Excitation with the WG filter having spectral range of 510 nm-550 nm emitted fluorescence in the 570-650nu range.

The epifluorescent microscopic screening of the cytogenetic slides 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 grey a phase contrast like effect.

Example 16 Fluorescent colour emitted The hues of colours emitted were noted in the areas where only dye was present and at places where some specimens were present. The excitation spectral range and the emitted fluorescence strictly followed the Stoke's law (Table 3).

Example 17 Microphotography of the slides with the dye used as epifluorescence microscopy stain The microphotography of emitted fluorescence in the areas of slides without cells and with specimen cells, under WU 330 nu -385 nu range, WB 450 nm -480 nm range, WG 510 rim-550 nm range and Bright field was done by Kodak film of 400 ASA speed with an exposure varying from 50 to 60 seconds. The results are shown in Figs 11-24.

Example 18 Stability Check The said dye is stable and remains active at room temperature and it remains like that up to 120 degree Centigrade and it was proved by no alteration of the spectral property after such treatment. The compound retained its stability for about a year without any contamination or chemical decay. The said marine dye did not undergo photolysis after light treatment. So the said marine dye does not require stabilizing agents.

Example 19 Pesticidal effect The compound was toxic to insects. It showed toxicity to the insects like ants. The filter paper soaked in the dye was left unattended on the workbench. Next day it was noticed full of dead ants.

Example 20 The dye was tested upon cell lines and activity observed Example 21 Staining with the Dye The fixed tissue with glacial acetic acid and methanol from different sources were taken on the slide and the dye solution was added to it without pre-treatment. It was observed that different parts of the cell were taking dye solution differently. The nucleus got stained because of the staining of arginine and lusine rich proteins present in the nucleus (Ex histone). The other cell organelles also got stained. As the followed marine dye is staining the proteins of chromosome it has added value in studying karyotype of the cell.

Example 22 The bioactive extract of the dye was taken in a microfuge tube and kept at-20 degree centigrade and seen in the frozen state under UV light. In another experiment the later dipped In the dye solution was held at -20OC and observed under UV transilluminator.

Example 23 : The extract was used as veterinary remedy for killing ticks/fleas of dogs. 1 : 200 times dilutions of the crude extract killed ticks and fleas in less than 40 seconds.

Table 1 (a) Structural analysis of the dye by chemical method for presence/absence of Quinonoid ring, phenolic and amine group.

Experiment Observation Inference 2 mg/ml of water sample + No decolouration Quinonoid ring absent p-marcaptoethanol 2 mg/ml of water sample + Purple colouration Phenolic ring is present neutral FeClg 2 mg/ml of water sample + No precipitate formed No aromatic amine group 0.1 N HCI + Na N02 in cold present added to alkaline solution of Beta-naphthol (Diazotization and then Beta-naphthol addition) NOTE: The tests which gave negative results were repeated by taking solution of higher concentration.

Elemental Analysis of the fluorescent dye :

1. Carbon, % 8. 3629 2. Hydrogen, 1.7430 % 3. Nitrogen, 0.8368 % 4. Sulphur, 9.4521 % Table 2 Structural analysis of the dye by chemical method for checking its proteinaceous/non- proteinaceous nature and presence/absence of the reducing sugar.

Experiment Observation Inference Heat a conc. Solution (5-10 No precipitation or Non-proteinaceous mg/ml) coagulation compound 2 mg/ml of water sample No colour change Quinonoid ring is absent beta marcaptoethanol NOTE: The tests which gave negative results were repeated by taking solution of higher concentration.

Table 3 The colour of the different coloured fluorescence of the dye used as stain when excited with different wavelength cubes of the Olympus epifluorescence microscope.

Name of the Excitation range of Emission range of *Colour of the fluorescent cube as the cube Fluorescence fluorescence given in the catalogue WU330-385 nm380-400 nmBlue WB 450-480 n 550-570 nm Yellow WG 510-550 nm 600-650 nm Orange Bright field Transmitted light Visible range Grey *NOTE: The microphotographs of these colours gave hues of the adjacent spectrum. For example, blue came as greenish hue, yellow came as greenish yellow, Orange came as reddish orange. Whereas while observing the colours were clear blue, yellow and orange fluorescence.

ADVANTAGES OVER THE PRESENT MARKETED DYES 1. The dye is non-radioactive as it is a dye from a natural source and not synthetic.

2. This dye in its single form is equivalent to three different synthetic fluorochromes giving same emission of fluorescent colours.

3. 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.

4. Being non degradable in quality of fluorescence for longer durations, it does not require refrigeration while exporting. The presently marketed fluorescent dyes exported under refrigeration equivalent to-20 degree centigrade.

5. Unlike the earlier known Green Fluorescent protein (GFP) from a marine jellyfish, our dye is not a reporter gene. The results are direct. GFP absorbs blue light at 395 nm and with a minor peak at 470 nm emits green light. Our dye emits 3 fluorescent colours and at three different fluorescent wavelengths. The dye is soluble in water-so can be used in components where water-soluble dyes are needed. The dye is insoluble in the organic solvents like ethanol, methanol and acetone.

6. The dye is negatively charged.

7. The dye has a pH of 6.5, which is almost neutral and hence will not affect final properties of pH drastically in compositions.

8. The dye is non-proteinaceous in nature so non degradable under natural conditions.

9. The dye has nature of a bio-surfactant so can be used in soaps and toiletry compositions.

10. The dye has anti-microbial qualities 11. The dye emitted these fluorescence colours even at a dilution range of 1 : 40000 times (i. e. I gm powder of dye dissolved in 40 litres of ultrapure water). The fluorescence of the extract persisted even after at least lyear at the room temperature. These multicoloured emissions of the dye at different wavelengths of excitations are comparable to the fluorochrome microscopic stains already in the market.

12. The blue coloured fluorescence of the present dye is comparable to the emission of same colour by DAPI fluorochrome at the same wavelength excitation, used as

components of the non-radioactive labelling kits of biochemistry, cell biology, immunochemistry, and molecular biology.

13. The blue coloured fluorescence of the present dye is also comparable to the emission of colour by Hoechest 33258 used as components of the non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.

14. The blue coloured fluorescence of the present dye is also comparable to the emission of colour by Hoechest 33342 fluorochrome at the same wavelength excitation used as components of the non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.

*15. The yellow coloured fluorescence of the said dye in the visible range is comparable to the same coloured emissions of Acridine orange used as components of the non radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.

16. The yellow coloured fluorescence of the said dye in the visible range is comparable to the same coloured emissions of auramine used as components of the non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.

17. The yellow coloured fluorescence of the said dye in the visible range is comparable to the same coloured emissions of FITC used as components of the non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.

18. The orange coloured fluorescent emission is comparable to the orange fluorescence colour of Propidium Iodide fluorochrome used as components of the non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.

19. The orange coloured fluorescent emission is comparable to the orange fluorescence colour of Rhodamine fluorochrome used as components of the non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.

20. The orange coloured fluorescent emission is comparable to the orange fluorescence colour of TRITC fluorochrome used as components of the non-radioactive labelling

and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.

21. 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.

22. The said single dye has characteristics of at least 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.

Under ordinary light of microscope the hues of greys produce a phase contrast effect, which is useful in rapid screening of cytogenetical, cytological, and histochemical slides and save expenses on the extra phase contrast accessory component of the microscope. The fluorescence colour emissions follow Stoke's law of fluorescence.

23. The microphotographs with Kodak film rolls show hues of the adjacent colour emission wavelengths. When blue colour fluorescence under the epifluorescence microscope is seen in the microphotograph the hues of green also shows.

24. The microphotographs with Kodak film rolls show hues of the adjacent colour emission wavelengths. When yellow colour fluorescence is seen under the epifluorescence microscope in microphotograph the hues of green also shows. When orange fluorescence colour is seen under the epifluorescence microscope in microphotograph the hues of red also shows.

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

26. The dye can be used for the preparation of polyvinyl chloride film that exhibits fluorescent colours. It also can be used in fluorescent colours in variety of paints, inks, and textiles.

27. The dye can be used in compositions of fluorescent dye for bleaching and brightening polymer. The dye can be used in leak detection with a full spectrum fluorescent dye. It can also be used in automated chemical metering system. It can also be used to mark the location of crashed aircrafts, life crafts, and equipment for example rockets. Further it can be used in under sea probes. The dye can be used in photo chemotherapy of skin cancers.

28. The dye can be used as chromatophore sunscreen component of cosmetics creams and lotions.

29. The water miscible quality of the dye can make it easily miscible in moisturizers. It can be used as fluorescent in situ hybridisation application kit component for molecular diagnostics. It can also be used as a component of the non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology for labelling of DNA, RNA, Proteins and enzymes, immunofluorescent detections, counterstain of DIG-labelled oliogonucleotide probes and anti-DIG Fab-fragments, single and multiple cell quantitative fluorescence in flow cytometry, fluorochrome stains for epifluorescence microscopy.

30. The dye can be used for a quick check of bio-contamination in the health food industry, cosmetic industry, pharmaceutical and chemical industries, for rapid estimations of bio-contaminants in laboratory cultures, for a rapid check of biopollutants under field conditions. It can also be a competitive inhibitor of cholinesterases.

31. The dye can be used in anti-microbial compositions.

32. The dye can be used as a bio-surfactant in toiletry compositions.

33. The dye can be used natural colorant A bioactive composition of the marine dye in the ratio of 1: 40000 in ultrapure water to obtain fluorescence of three colours at three different wavelengths and a phase contrast effect under transmitted light.

34. Purification of the dye can be done in 250 ml of 50% ethanol crude extract when evaporated on a water bath at 80 degree centigrade for 5 minutes to purify provide 2.5g of purified dye in a powder form. 2mg/10 ml solution composition is used for spectrophotometry Structural analysis of the dye using chemical method using concentration of 10 mg/ml was used. A bioactive composition of the dye in the ratio of 1: 40,000 times dilutions with water as the binder gives fluorescence of three colours at three different wavelengths.

Claims (54)

1. CLAIMS : 1 A bioactive extract obtained from a marine organism and useful as a natural fluorescent dye having the following characteristics: i. depolarisation by a reducing agent, ii. not a synthetic compound, iii. crude extract of the dye is yellowish green in colour, iv. purified dye being reddish brown coloured powder when seen with the naked eye in the daylight, v. under tube light some hues of green are emitted, vi. amorphous in nature, vii. soluble in water, viii. insoluble in the organic solvents like ethanol, methanol and acetone, ix. is negatively charged, x. has a pH of 6. 5, xi. presence of a phenolic group, xii. absence of a quinoid ring, xiii. absence of aromatic amine groups, xiv. non-proteinaceous in nature, xv. reducing sugar is absent, xvi. dye has nature of a biosurfactant, xvii. dye also showed antimicrobial qualities and when antimicrobial assay was performed, showed zone of inhibition, xviii. pigment cum dye is a fluorescent dye and emits fluorescence when excited with different wavelengths of UV and visible spectral ranges on a spectrophotometer, xix. UV, visible spectroscopy from 300nm-700nm and the peaks are marked at

   379 nm and 439nm wavelengths, xx. UV, visible spectroscopy from 250 nm-350nm and the peaks are at 272 nm and 299 nm wavelengths, xxi. fluorescent spectroscopy in the UV and visible spectra, when excited with UV

   270 nm wavelength the fluorescence is emitted in the 324nm-380nm range

   which comes under the UVA wavelength range of ultraviolet rays of the sunlight, xxii. with excitation wavelength 450nm in Fluorescent spectroscopy the fluorescence emission occurred at 500nm-580 nm with maximum intensity, xxiii. with excitation wavelength 540nm in Fluorescent spectroscopy, the fluorescence emission occurred at 500nm-620 nm with maximum intensity, xxiv. with excitation wavelength 555 nm in Fluorescent spectroscopy, the fluorescence emission occurred at 575 nm-620 nm with maximum intensity, xxv. physical checking of Whatman Filter no. 1 dipped with dye concentration 1: 40000 dilution under UV transilluminator and Gel Documentation system with UV bulbs of 260nm-280nm range emit bluish green hue colour of fluorescence, xxvi. emits three different coloured fluorescence at 3 different wavelengths of the UV and visible ranges of the fluorescent cubes of an epifluorescence microscope, xxvii. fluorescence blue colour emission occur in the 380nm-400 nm range of UVA

   when excited under ultra violet cube WU-330 nm-385 nm excitation range, xxviii. fluorescence yellow colour emission occurs in the 500nm-570nm range when excited under WB cube of 450nm-480 nm excitation range, xxix. fluorescence orange colour emission occurs in the 570 nm-650nm range when excited under WG cube of 51 Onm-550 nm excitation range, xxx. the dye emits hues of greys under the ordinary transmitted light bulb of the epifluorescence microscope when seen under 10X objective, xxxi. the dye emitted these fluorescence colours even at a dilution range of 1: 40000

   times (i. e. l gin powder of dye dissolved in 40 litres of ultrapure water), xxxii. the fluorescence of the extract persisted even after at least 1 year at the room temperature, xxxiii. the fluorescence of the dye is highly photostable and does not get deteriorated by long exposures to direct light, and xxxiv. the fluorescence of the dye does not change even when frozen at -20OC, a temperature at which the molecules are unable to attain the energy necessary for activation like in extracts from luminescent organisms.
2. 2. A dye as claimed in claim 1 wherein the dye is obtained from marine organisms such as Holothuria scabra occurring in intertidal, submerged, shallow and deep waters, usually abundant in shaded areas such as alcoves, crevices, ledges, overhangs, rocky and sandy habitats; dull to bright coloured with or without exo and endoskeleton, sessile, sedentary drifters, nektonic with varied swimming power usually nocturnal in habit, labile to active predation.
3. 3. A dye as claimed in claim 1 wherein the multicoloured emissions of the dye at different wavelengths of excitations are comparable to the fluorochrome microscopic stains already in the market.
4. 4. A dye as claimed in claim 1 wherein the blue coloured fluorescence of the present dye is comparable to the emission of same colour by DAPI fluorochrome at the same wavelength excitation, used as components of the non-radioactive labelling kits of biochemistry, cell biology, immunochemistry, and molecular biology.
5. 5. A dye as claimed in claim 1 wherein the yellow coloured fluorescence of the said dye in the visible range is comparable to the same coloured emissions of Auramin used as components of the non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
6. 6. A dye as claimed in claim 1 wherein the yellow coloured fluorescence of the said dye in the visible range is comparable to the same coloured emissions of FITC used as components of the non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
7. 7. A dye as claimed in claim 1 wherein the orange coloured fluorescent emission is comparable to the orange fluorescence colour of Propidium Iodide fluorochrome used as components of the non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
8. 8. A dye as claimed in claim 1 wherein the orange coloured fluorescent emission is comparable to the orange fluorescence colour of Rhodamine fluorochrome used as components of the non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
9. 9. A dye as claimed in claim I wherein the orange coloured fluorescent emission is comparable to the orange fluorescence colour of TRITC fluorochrome used as

   components of the non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology.
10. 10. A dye as claimed in claim 1 wherein the dye is stable at the room temperature and has a long shelf life.
11. 11. A dye as claimed in claim 1 wherein the molecular and radioactive kits of the said dye can be exported at the room temperatures.
12. 12. A dye as claimed in claim 1 wherein the single dye has characteristics of at least one hundred different fluorochromes namely (DAPI, Hoechest 33258, Hoechest 33342, FITC, acridine orange, auramine, Rhodamine, TRITC, and propidium iodide, etc. ) which are now in the market.
13. 13. A dye as claimed in claim 1 wherein it can be used in all applications where presently Phycobiliproteins are used as unlike them the dye does not undergo loss in fluorescence upon freezing.
14. 14. A dye as claimed in claim 1 wherein under bright field of fluorescent microscope when seen under 10 X objective the hues of bluish greys produce a phase contrast effect which is useful in rapid screening of cytogenetical, cytological, and histochemical slides and save expenses on the extra phase contrast accessory component of microscope.
15. 15. A dye as claimed in claim 1 wherein under 100 X oil immersion objective of an ordinary transmitted light microscope the proteins of yolk, nucleoplasm and chromatin of actively dividing cleavage cells show different colours of staining in the hues of brownish yellow for former, yellow for the latter and dark blue for the last cell component. This can be useful in rapid bioassays of effect can be seen on the various histochemical components of the cells.
16. 16. A dye as claimed in claim 1 wherein the fluorescence colour emissions follow Stoke's law of fluorescence.
17. 17. A dye as claimed in claim 1 wherein the microphotographs with Kodak film rolls shows hues of the adjacent colour emission wavelengths such as blue colour fluorescence under the epifluorescence.
18. 18. A dye as claimed in claim I wherein the microphotographs with Kodak film rolls shows hues of the adjacent colour emission wavelengths like when seen yellow

    colour fluorescence under the epifluorescence microscope in microphotograph the hues of green also comes.
19. 19. A dye as claimed in claim 1 wherein when seen orange fluorescence colour under the epifluorescence microscope in microphotograph the hues of red also comes.
20. 20. A dye as claimed in claim 1 wherein the cytogenetic slides seen under all fluorescence gives a counterstain effect of cells and cell components verses the background colour where no specimen but only dye is present.
21. 21. A dye as claimed in claim I wherein the dye is diluted with water in the ratio 1: 10,000 times and this gives fluorescence of three colours at three different wavelengths.
22. 22. A dye as claimed in claim 1 wherein the dye is diluted with water in the ratio 1: 40,000 times and this gives fluorescence of three colours at three different wavelengths.
23. 23. A composition comprising a bioactive extract obtained from the marine sea cucumber Holothuria scabra together with conventional additives and useful for the preparation of flexible polyvinyl chloride film that exhibits fluorescent colours.
24. 24. 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.
25. 25. A composition comprising a bioactive extract obtained from the marine sea cucumber Holothuria scabra together with conventional additives and useful in detection of leaks.
26. 26. A composition comprising a bioactive extract obtained from the marine sea cucumber Holothuria sabra together with conventional additives and useful in undersea probes.
27. 27. A composition comprising a bioactive extract obtained from the marine sea cucumber Holothuria scabra together with conventional additives and useful in photo chemotherapy of skin cancers.
28. 28. A cosmetic composition comprising a bioactive extract obtained from the marine sea cucumber Holothuria scabra together with conventional additives.
29. 29. A composition comprising a bioactive extract obtained from the marine sea cucumber Holothuria sabra together with conventional additives and useful as a fluorescent molecular probe in situ hybridisation kits for molecular diagnostics.
30. 30. A composition comprising a bioactive extract obtained from the marine sea cucumber Holothuria sabra together with conventional additives and useful as a component of non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry and molecular biology.
31. 31. A composition comprising a bioactive extract obtained from the marine sea cucumber Holothuria scabra together with conventional additives and useful in immuno fluorescent detections.
32. 32. 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-labelled oliogonucleotide probes and anti-DIG Fab-fragments.
33. 33. A composition comprising: a bioactive extract obtained from the marine sea cucumber Holothurie scabra together with conventional additives and useful in single and multiple cell quantitative fluorescence in single and multicolour flow cytometry applications.
34. 34. A composition comprising: a bioactive extract obtained from the marine sea cucumber Holothuria scabra which can be used in experiments where various applications of fluorescent dyes are needed to be performed at field stations situated at subzero degree temperature areas.
35. 35. 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.
36. 36. 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.
37. 37. A composition comprising a bioactive extract obtained from the marine sea cucumber Holothuria scabra together with conventional additives and useful for rapid estimations of bio-contaminants in laboratory cultures.
38. 38. A composition comprising a bioactive extract obtained from the marine seacucumber Holothuria scabra together with conventional additives and useful for a rapid check ofbio-pollutants under field conditions.
39. 39. A composition comprising a bioactive extract obtained from the marine sea cucumber Holothuria sabra together with conventional additives and useful as a competitive inhibitor of cholinesterases.
40. 40. A composition comprising a bioactive extract obtained from the marine sea cucumber Holothuria scabra together with conventional additives and useful in antimicrobial compositions.
41. 41. A composition comprising a bioactive extract obtained from the marine sea cucumber Holothuria scabra together with conventional additives and useful as a biosurfactant in toiletry compositions.
42. 42. A composition comprising a bioactive extract obtained from the marine sea cucumber Holothuria sabra together with conventional additives and useful in pesticidal compositions.
43. 43. A composition comprising a bioactive extract obtained from the marine sea cucumber Holothurie scabra together with conventional additives and useful as a natural colorant.
44. 44. A bioactive composition containing an extract obtained from the marine sea cucumber Holothuria sabra in the ratio of 1: 40000 in ultrapure water to obtain fluorescence of three colours at three different wavelengths.
45. 45. A bioactive composition containing an extract obtained from the marine sea cucumber Holothuria sea to obtain a phase contrast and histochemical counter stain effect for different biochemical constituents of cells under transmitted light.
46. 46. A skin care composition containing the dye together with physiologically and cosmetically acceptable vehicle such as diluent, dispersant or carrier.
47. 47. A dye as claimed in claim 1 useful for: (a) preparation of flexible polyvinyl chloride film that exhibits fluorescent colours ; (b) use of fluorescent colours in variety of paints, inks, textiles; (c) a composition of fluorescent dye for bleaching and brightening polymer; (d) leak detection with a full spectrum fluorescent dye; (e) use in automated chemical metering system; (f) to mark location of crashed air-crafts, life crafts, and equipment for example rockets; (g) under sea probes; (h) UVA is used in photo chemotherapy of skin cancers; (i) chromatophore sunscreen component of cosmetics creams and lotions; (j) the water miscible quality of the dye can make it easily miscible in moisturizers; (k) fluorescent in situ hybridisation application kit component for molecular diagnostics; (I) component of the non-radioactive labelling and detection kits of biochemistry, cell biology, immunochemistry, and molecular biology for labelling of DNA, RNA, Proteins and enzymes; (m) immunofluorescent detections; (n) counter stain of DIG-labelled oligonucleotide probes and Anti-DIG Fab fragments; and (o) single and multiple flow cytometry applications; (p) fluorochrome stains for epifluorescence microscopy; (q) for a quick check of bio-contamination in the health food industry, cosmetic industry, (r) pharmaceutical and chemical industries;

    (s) for rapid estimations ofbio-contaminants in laboratory cultures ; (t) for a rapid check ofbio-pollutants under field conditions ; (u) competitive inhibitor of cholinesterases ; (v) in anti-microbial compositions; (w) as a bio-surfactant in toiletry compositions; (x) a natural colorant; (y) a bioactive composition of the marine dye in the ratio of 1: 40000 in ultrapure water (z) to obtain fluorescence of three colours at three different wavelengths and a phase contrast effect under transmitted light;

    (aa) a dye for various fluorescent applications to be performed in areas of sub zero temperatures.
48. 48. A process for extraction of a natural fluorescent dye from Holothuria scabra sea cucumber, which comprises the steps of : a) collecting the material from seashore, changing of seawater and maintenance in the laboratory tanks without any mechanical aeration overnight, b) freezing the animals at -20OC, c) thawing the animal to obtain the pigment; and d) repeating the steps (b) and (c) 3-4 times for removal of pigment and, if desired, purify the pigments.
49. 49. A process for extraction of a natural fluorescent dye from Holothuria scabra sea cucumber which comprises the steps of extraction of the pigment from the skin of the sea cucumber Holothuria scabra by osmotic shocks preferably with adding 50% ethanol to the ultrapure water on first time, and repeating this at least 4 times to obtain the dye.
50. 50. A process as claimed in claim 48 wherein the dye is diluted with water in the ratio

    1 : 40,000 times and this gives fluorescence of three colours at three different wavelengths.
51. 51. A process as claimed in claim 48 wherein 250 ml of 50% ethanol crude extract when evaporated on a water-bath at 80 degree centigrade for 5 minutes to purify provide 2.5g of partially purified dye in a powder form.
52. 52. A process as claimed in claim 48 wherein the dye is diluted with water in the ratio 1: 10,000 times and this gives fluorescence of three colours at three different wavelengths.
53. 53. A process as claimed in claim 48 wherein the physical characteristics of the dye are assessed by taking 2mg/10 ml solution for spectrophotometry.
54. 54. A process as claimed in claim 48 wherein the dye was used at a concentration of 10 mg/ml for analysis of the dye using chemical method.