JP2005538032A - Fluorescent natural pigment derived from marine organisms - Google Patents

Abstract

本発明は、Ｓｉ−Ｏ−Ｒ型（Ｒは、有機部分である）の化学構造を有する、海洋生物のハマジナマコ（Holothuria scabra）由来の、新規化合物多重蛍光性天然色素を提供する。ケイ酸マトリックスが、前記化合物の必須部分であり、動物の代謝において役割を果たしており、種々の適用を有する前記化合物を含む組成物も提供する；また、海洋生物（ハマジナマコ（Holothuria scabra））由来の、新規な多重蛍光性天然色素化合物の抽出、精製および特徴づけの方法も提供する。【Task】
[Solution]
[Chemical 1]

The present invention provides a novel compound multi-fluorescent natural dye derived from the marine organism Holothuria scabra having a chemical structure of the Si-O-R type (R is an organic moiety). A silicate matrix is an essential part of the compound and plays a role in animal metabolism and also provides a composition comprising the compound with various applications; also from marine organisms (Holothuria scabra) Also provided are methods for the extraction, purification and characterization of novel multiple fluorescent natural dye compounds.

Description

TECHNICAL FIELD The present invention relates to a novel organosilicon Si—O—R type compound, which comprises a marine species belonging to the genus: Echinodermata, Class: Sea cucumber, Eye: Aspidochirota, Family: Holothuroidae It is a multiple fluorescent natural pigment purified from an inner wall extract of invertebrate sea cucumber (Holothuria scabra). The compound is a polysaccharide fluorescent dye having a phenolic phosphor moiety, and is bound to a silicon matrix around it by sulfate bonds. This silicon moiety is an essential part of the nuclear molecule and contributes to animal metabolism. The compound is rich in sulfur.

  The present invention also provides methods for the extraction, purification and characterization of novel compounds and multiple fluorescent dyes from living marine organisms, particularly sea cucumbers.

  The present invention also disclosed for the first time the chemical structure of a novel fluorescent compound in which silicon is an essential part of the organic molecule and the phenolic form of its phosphor. Furthermore, the unique properties of the compounds and the characteristics of the dyes are provided and their advantages are disclosed. We have also described the industrial utility of novel fluorescent compounds as multiple fluorescent natural dyes for non-radioactive labeling for fluorescent in situ hybridization, flow cytometry, immunoassay and fluorescence microscopy. Companies that trade high molecular weight fluorescent compounds (which have been sought after due to some special requirements, but were not available) in the market for fluorescent molecular probes. By providing the company with an alternative to low molecular weight fluorescent dyes by providing only to that company, and also by utilizing only the phosphor portion thereof, has another utility for the company. Several derivative dyes have been produced that have the desired properties for high and low molecular weight and fluorescent probes for single and multiple color applications. In addition, the compounds can be readily incorporated ingredients in the composition for the cosmetic industry, in particular sunscreens, and can be drugs in pesticides and animal therapeutics.

See Prior Art Silicon has been described to play an active role in plant and animal development. F. C. Lanning, FC (“The encyclopaedia of the chemical elements”, by CA Hampel, Reinhold Book Corporation. Inc, New York, a subsidiary of Chapman-Reinhold, pages 647, 1968. According to the year, silicon may play an important and essential role on the origin of life on earth. The lower forms of life, such as diatoms, often use silicic acid in the skeletal structure. He proposes that Si-O-R type bonds may occur in living organisms. However, so far no reports have been available in the prior literature that silicic acid becomes an integral part of the organic moiety and may act as a matrix to render the compound fluorescent. Wannagat describes that the silicon content of living organisms decreases with the complexity of biogenesis. The ratio of silicon to carbon is 250: 1 in the extraterrestrial layer, 15: 1 in humus, 1: 1 in plankton, 1: 100 in ferns, and 1 in mammals. : 5000 (quoted from wysiwyg: // 34 / http: //www.pbs.org/wgbh/pages/frontline/implants/corp/history.html). The author reports that silicon plays an important role in the growth of hair, nails, bones and wings, but that role is not fully understood. At the fracture site, the silicon content is increased by a factor of 50 in the collagen network.

  Most organosilicon compounds on the market are synthetic. Silicon is a comprehensive description for a fully synthetic polymer containing a repetitive Si-O backbone. An organic group linked to a silicon atom via a silicon-carbon bond is defined as a collection of silicones. They are sold on the market as fluids, emulsions, compounds, lubricants, resins and elastomers or rubbers and are also used in cosmetic plastic surgery (wysiwyg: // 34 / http: //www.pbs. (quoted from org / wgbh / pages / frontline / implants / corp / history.html). However, for silicon compounds in living organisms, no information is available in the literature that silicic acid is an essential part of organic molecules and also has the characteristics of single or multiple fluorescent dyes. Most of the dyes currently available on the market are synthetic. Stainfile-Dye A is given a dye index of 264 dye. Of these, 258 are synthetic and only 6 are natural pigments (http://members.pgonline.com/-bryand/dyes/dyes.htm). The production of synthetic dyes often requires the use of strong acids, strong alkalis and heavy metals as catalysts at elevated temperatures. This makes these methods and wastewater a problem of environmental degradation. The dye industry is continually looking for cheaper, more environmentally friendly routes to existing dyes (Hobson and Wales, 1998, “Green Dyes”, Journal of the Society of Dyers and colorists (JSDC), 1998, 114, 42-44). All of the available dyes are not fluorescent. The Bitplane product displays a list of 123 fluorescent dyes and their excitation and emission spectra on the market (http://www.bitplane.ch/public/support/standard/fluorochrome.htm) . Fluorescent dyes are widely used for labeling molecular probes to localize biological structures by fluorescence microscopy, for example, immunoassays, nucleotides and oligonucleotides for in situ hybridization studies Are widely used in the labeling of proteins, binding to polymeric microspheres, and staining of cells used in imaging studies. The dyes are also used for selective destruction of cells, for example in photochemical therapy techniques (RP Haughland, RP and HC Kang, HC US Pat. No. 4,774,339, effective 27 September 1988; RP Holland (RP) and HC Kang, HC, US Pat. No. 5,248,782, entered into force on September 28, 1993).

Fluorescence is a phenomenon in which atoms or molecules emit radiation in the process of changing from a high electronic state to a low electronic state. It follows Stoke's law, according to which the wavelength of the fluorescent radiation is always longer than the wavelength of the excitation radiation. The fluorescence process is completely different from phosphorescence and bioluminescence. The term “fluorescence” is used when the interval between the act of excitation and the emission of radiation is very small (10 ⁻⁸ −10 ⁻³ seconds). In phosphorescence, the time interval between absorption and emission varies from 10 ⁻³ seconds to several hours (R. Norman Jones, 1966, “The encyclopaedia of chemistry) ", 2nd edition, 1966, 435-436). Bioluminescence is the term used for the light produced as a result of chemical reactions occurring in specific cells at specific times within the body of a living organism.

  A number of fluorescent dyes are described in the "Handbook of Fluorescent probes and Research Chemicals" (Richard P. Haughland, 6th edition, US publication, 1996). Is reported. On pages 1-6 of the same book, Ian D. Johnson (1996) described the process of fluorescence and certain molecules (usually polyaromatic hydrocarbons or heterocycles) that he called fluorophores or fluorescent dyes. Details of the detection method in the ring) are described. Currently, the most widely used fluorescent dyes are fluorescein and fluorescein-based, and BODIPY dyes and their derivatives.

  The author deals with the shortcomings of all these dyes and describes the choice of dye characteristics. Many derivatives of the fluorescent dyes and their synthesis are disclosed in US patents (RP Haughland, RP and HC Kang, US Pat. No. 4,774,339, 1988). Effective September 27; RP Holland (RP) and HC Kang (Kang, HC), US Pat. No. 5,248,782, September 28, 1993; RP Holland (RP) and HC Kang ( Kang, HC), US Pat. No. 5,187,288, published February 16, 1993; RP Haughland, RP and HC Kang, HC, US Pat. No. 5,274,113, December 28, 1993; HC Kang, HC and RP Holland, US Pat. No. 5,433,896, July 18, 1995; HC Kang, HC and RP Holland. (Haughland, RP), rice (National Patent No. 5,451,663, published on September 19, 1995). Rosenblum Barnett B, Spurgeon S, Lee Linda G, Benson Scott C and Graham Ronald J J) reports that 4,7-dichlororhodamine dye is useful as a molecular probe in International Patent Application No. W00058406 (published on Oct. 5, 2000). R. Norman Jones describes good phosphors in "the encycopaedia of chemistry, second edition, 1966, 435-436". According to him, fluorescent molecules are used for absorption and shielding mechanisms of excitation energy, in order to save the excitation energy from disappearing too quickly into the oscillating motion before the fluorescence retardation effect occurs. Must have a good chromophore system. He also states that the relationship between molecular structure and compound fluorescence is not well understood, but certain groups exist and their presence is related to fluorescence. For example, in organic molecules, the presence of phthalein and aromatic structures such as anthracene and naphthacene is particularly associated with bright fluorescence. Inorganic compounds are liquid and solid, rarely emit strong fluorescence, and fluorescence is often modified by a small amount of impurities.

  The inventors of this patent application, Goswami USHA and Ganguly Anutosh, have already filed patent applications for natural fluorescent dye extracts from marine invertebrates (US patent application no. 09/820, 654). This relates to a crude extract from sea cucumber sea cucumber (Holothuria scabra), which has fluorescent properties at three different wavelengths when excited in different UV and visible regions of the spectrum of light. The invention also provides a method for the extraction, purification and characterization of this new pigment, which is a particularly purified natural pigment derived from sea cucumber. Usefulness of the dyes as epifluorescence dyes and non-radioactive fluorescent dyes useful for labeling molecular probes for in situ hybridization studies is as follows: In addition to several other properties, it is described. In this application, details of the prior art are described for said pigments, synthetic pigments and natural pigments from terrestrial plants and microorganisms (US Pat. No. 4,452,822, June 1984). Released 5th; Inventor-Shrikhande, Anil J; US Patent No. 5,321,268, June 14, 1994, Crosby David A and Exome Philip A (Ekstrom Philip A); U.S. Patent No. 5,405,416, published April 11, 1995, author Swinton; Robert J, U.S. Patent No. 5.858,761 Published on January 12, 1999, inventor Tsubokura et al., US Patent No. 5,902,749 on May 11, 1999, inventor Lichtwardt et al. No. 5,908,650, published Jun. 1, 1999, Inventor Lenoble et al., U.S. Patent No. 5,920,429, published Jul. 6, 1999, Burns et al. U.S. Pat. No. 5,935,808, Hirschberg et al., August 10, 1999; U.S. Pat. No. 5,989,135, November 23, 1999, inventor Welch David. Emmanuel (Welch; David Emanuel); U.S. Patent No. 6,055,936, effective May 2, 2000; Collin; Peter Donald; U.S. Patent No. 6,056,162; May 2, 2000; Leighley; Kenneth C .; US Patent No. 6,103,006, August 15, 2000, DiPietro; Thomas C. 6,110,566, August 29, 2000; White et al .; 6,140,041, October 31, 2000, LaClair; James J. 6,165,384, Cooper et al., Dec. 26, 2000; 6,180,154, Jan. 30, 2001, Wrolstad et al., European Patent No. EP 0206718, published Dec. 30, 1986, inventor Kramer.・ Cramer Randall J; IE901379, January 30, 1991, Lee Linda G; Mize Patrick D; WO9010044., July 7, 1990. Swinton; Robert J; AU704112, published October 7, 1997, inventor Burns David M; Pavelka Lee A; June 24, 1999 Daytimer Thomas Dr. (Weimer Thomas DR.) DE 19755642; WO99389919, September 28, 1999, Laclair James J; October 5, 2000, Rosenblum Barnett B et al. WO99389916, August 15, 2000, inventor DiPietro; Thomas C; WO9920688, August 29, 2000, inventor Pavelka Lee, et al .; August 29, 2000; WO 9920688, inventor White et al.).

  Various uses of fluorescent dyes in molecular biology research, industrial applications and life-saving devices have also been described. Collin, PD, June 23, 1998, March 2, 1999 and November 16, 1999 (each US Pat. No. 5,770,205, No. 5) , 876,762 and 5,985,330) describe the therapeutic properties of various body parts of sea cucumbers. All these references are also relevant to the present invention.

  In this patent, the inventors have taken a different approach. In this invention, the inventors purify chemical compounds and fluorescent dyes, which are unique in their structure and initially showing eight colored emissions. The chemical structure of the compound was derived from sea cucumber and was disclosed for the first time. Both the product and the source are new. The phosphor portion of the compound is phenolic, and both the entire compound and only the phosphor have the properties of a natural fluorescent dye. Unlike currently available dyes, this compound is not any synthetic derivative of dyes currently sold on the market. It is a completely new organosilicon Si—O—R type compound and was first purified and disclosed in this invention. This novel fluorescent polysaccharide extraction, purification and characterization method from sea cucumber inner wall extracts was also novel and first disclosed. The fluorescent nature of the disclosed polysaccharide is also novel and its use as a natural multiple fluorescent dye for molecular probes is also novel. This is the first time that the compound is of the Si-O-R type and the nuclear organic molecule is bound to the silicon matrix around it by sulfate bonds. The disclosure that the silicon matrix forms an essential part of the compound and plays a role in sea cucumber metabolic activity is also novel.

  In another aspect, the present invention provides novel natural compounds to interested molecular probe companies and molecular chemical companies, which are also polymeric fluorescent dyes and low molecular weight fluorescent dyes. Depending on the requirements, they can be provided to the company.

  In yet another aspect, the invention is that the compound is rich in sulfur and its various compositions exhibit utility as insecticides, pesticides and animal drugs.

  Unlike most other known fluorescent synthetic dyes, our dyes do not need to be mixed with another dye to obtain different fluorescent tones at different wavelengths in epifluorescence microscopy. After contact with the biomolecule, it emits fluorescence colored in 8 different colors at 8 different excitation wavelengths, which can have a variety of uses. It stains the cell membrane and emits red, blue, yellow and orange shades under different emission wavelengths of the fluorescence microscope. Furthermore, our dyes are essentially non-proteinaceous, very stable at room temperature for several months, and are not contaminated by microorganisms. Its fluorescent properties do not deteriorate at high and low temperatures, unlike some algae and luminescent organism extracts. This dye does not show a rapid quenching effect when the cell sample is exposed to light under a microscope.

  One important aspect of this fluorescent dye is to make compositions and kits for non-radioactive labeling and counterstaining of molecular probes. Its chemical structure has been disclosed, so that the dye manufacturing industry can be synthesized on a large scale. In different wavelength excitation, it produces an effect equal to the color spectrum color of at least 123 fluorescent dyes currently known on the market (see Bitplane product (fluorescent dye) on the Internet (http://www.bitplane.ch /public/support/standard/Fluorochrome.htm)).

  In yet another aspect, the dye emits light in a longer range when excited by high energy X-rays. It emits yellowish green fluorescence.

  Yet another aspect is the use as a fluorescent dye in epifluorescence microscopy, which is reported here for the first time as a marine natural dye. The dye produces a counterstaining effect of different cell components. With this application, various applications such as molecular diagnostics using fluorescent in situ hybridization technology, rapid diagnosis of biological contamination in tissue culture, industrial preparation, water property check in laboratory and field situations, Provides a simple and quick method for checking cytogenetic samples for use.

  Yet another aspect of the dye is its use as a component of a non-radioactive labeling kit for advanced molecular biology applications and as a drug in medical and animal preparations.

Objects of the present invention The main object of the present invention is to provide a method for the extraction, purification and characterization of said compounds and multiple fluorescent dyes from sea cucumber holothuria scabra.

  Another object of the present invention is to provide the chemical formula, molecular weight and structure of the purified compound for industrial application on its large scale.

  It is a further object of the present invention to provide compositions using pure compounds and fluorescent dyes obtained from sea cucumber body wall extracts.

  A further object of the invention is the analysis of the silicon matrix for its properties.

  A further object of the present invention is that the silicon matrix is an essential part of the nuclear organic molecule.

  Yet another object of the present invention is that the silicon matrix provides stability to the compound and the phosphor emits fluorescence to the compound.

  Still another object of the present invention is that the purified fluorescent compound is a polysaccharide. The nuclear organic molecules of the Si-O-R type compound are bound to the silicon matrix by sulfate bonds around them.

  Yet another object of the present invention is that the dye binds directly to its structure without the need for further spacers. Sulfate base serves as a spacer.

  Yet another aspect of the present invention is that the dye emits a different color after binding to the protein biomolecule.

  In yet another aspect, the present invention provides novel natural compounds to molecular probe companies and molecular chemical reagent companies of interest and provides them with high molecular weight fluorescent dyes. The dye can also be used as a low molecular weight by using only the phosphor moiety depending on the requirements.

  Yet another aspect of the invention is to make it clear that both the phenolic and quinonoid structures of the compounds are responsible for fluorescence.

  Yet another object of the present invention is that the silicate matrix plays a role in sea cucumber metabolic activity.

  Yet another aspect of the present invention is that the compound is rich in sulfur and its various compositions show utility as insecticides and pesticides.

  Yet another object of the present invention is to give the compound a chemical taxonomic name as phenolic sulfated silicated polysaccharide (PSSP-1).

  Yet another object of the present invention is to give the phosphor a chemical taxonomic name as 1-hydroxy, 2,5-di-methylaminobenzene (HDAB-1).

  Yet another object of the present invention is its application as a drug to inhibit connective tissue growth in the reconstruction of cartilage skeletal parts in mammals.

  Yet another object of the present invention is the usefulness of the compounds in sunscreen cosmetics.

  Yet another object of the present invention is that when a marine animal cell / larvae microscope slide is stained, the dyes fluoresce in 8 different colors, the pure dyes having different hues, The bound cell component is to have a different color.

  Yet another object of the present invention is that the dye emits green fluorescence even when excited by high energy rays such as X-rays.

  Yet another object of the present invention is to observe fluorescence and visible spectroscopic analysis and a range of emission wavelengths.

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

  Yet another object of the present invention is that the dye does not quench rapidly in the excitation light and no photobleaching occurs during the screening slide.

  Another object of the invention is that the dye is essentially non-proteinaceous and highly stable at room temperature for many years.

  Yet another object of the present invention is that its fluorescent properties do not deteriorate, even at extremely high and low temperatures.

  Yet another object of the present invention is to develop a kit for non-radioactive labeling of molecular probes and counterstains.

  Yet another object of the invention is the industrial use of said compounds for synthesizing derivatives of fluorescent dyes for flow cytometry, microarrays, immunoassays and some other molecular applications.

SUMMARY OF THE INVENTION Accordingly, the present invention provides novel natural organosilicon-type compounds and multiple fluorescent dyes obtained from sea cucumber holothuria scabra. The present invention also provides methods for extraction, purification and characterization of said compounds and multiple fluorescent natural dyes for fluorescent molecular probes, cosmetics, drugs and other industries. Furthermore, the present invention provides the entire chemical structure of a purified compound containing its phosphor and fluorescent dye moiety. The present invention also provides both the phenolic and quinonoid form of the compound responsible for its fluorescence. Based on chemical structure, the present invention also identifies PSSP-1 as “phenolic sulfated silicated polysaccharide”, “1” represents the first compound, and identification of compounds and highly commercially available fluorescent dyes. Some derivatives that provide a name for will follow this patent. Furthermore, the present invention provides the structure of the phosphor, its nomenclature, for example 1-hydroxy, 2,5-di-methylaminobenzene, and this dye has been named HDAB-1. This HDAB is associated with a given nomenclature, “1” which is the first several conjugates, and dyes based on this phosphor will follow.

  Furthermore, the present invention provides a composition of said compounded (cum) fluorescent dye to be tested as a molecular probe for marine animal cells, their antibacterial, insecticide and agrochemical activity. Said pigments have drug properties for animal and cosmetic applications.

Detailed Description of the Invention After much research, we have now identified, purified and structurally elucidated novel organosilicon-type compounds and novel multiple fluorescent natural dyes, Characterized. Said fluorescent dyes are obtained from marine animals, in particular invertebrates, more particularly from sea cucumber holothuria scabra. Sea cucumbers are in the following taxonomic positions: The sea cucumber is an echnoderm, a member of a group of animals whose body surface is thorned, including starfish and sea urchins.
Subworld: Metazoans Gate: Echinoderms Suba: Protists Class: Sea cucumber Subclass: Abuta
Eye: Azusa
Family: Holothuroidae
Genus: Sea cucumber
Species: Scabra
The compound is a polysaccharide and is a polysaccharide. This purified compound is a multiple fluorescent dye having a phenolic phosphor moiety. It is bound to the silicon matrix around it by sulfate bonds. This silicon moiety is an essential part of the nuclear molecule and plays a role in animal metabolism. The compound is rich in sulfur. The invention also provides a method for the extraction, purification and characterization of this new fluorescent compound from living organisms, especially sea cucumbers. The application also discloses for the first time the chemical structure of a novel compound in which silicon is an essential part of the organic molecule. The application also reveals both the phenolic and quinonoid structures of the compound that are responsible for fluorescence. The present application further provides the unusual properties of the compound and its advantages. The present invention also addresses the industrial applicability of the novel fluorescent compounds as dyes for non-radioactive labeling of molecular probes in various molecular biology, molecular probes and drug applications. Several chemical derivatives of the compounds can be made for a variety of molecular probes for different applications. Furthermore, the present invention provides a composition comprising an insecticide, an agrochemical and the dye for therapeutic use.

Accordingly, the present invention provides a novel multiple having the empirical formula C ₂₂ H ₄₈ O ₆₆ N ₂ S ₁₀ Si ₇ , having a molecular weight of 1915, and having the structural formula depicted in FIG. 18 or 19 of the accompanying drawings. A fluorescent dye compound is provided.

In an embodiment of the invention, the novel compound has the following characteristics:
i. The organic composition of the pure compound by elemental analysis shows carbon 8.572, hydrogen 1.739%, nitrogen 0.9449% and sulfur 9.457%;
ii. The atomic ratio for the element in the compound is the carbon: hydrogen: nitrogen: sulfur ratio shown by microanalysis = 22: 48: 2: 10;
iii. Subjecting the inorganic composition of the compound to atomic absorption photometry to estimate a silicon dioxide fraction of 12.58% and silicic acid;
iv. Silicic acid is an essential part of the compound;
v. The atomic ratio of silicon is 7 for every 22 carbon atoms;
vi. Further purification of the compound by ion exchange chromatography and confirmed that the absorbance peak-to-peak ratio at 291 and 451 is stable;
vii. The compound comprises a sugar unit;
viii. HPLC of the pure ionic form of the compound with a reverse phase C18 type analytical column eluting the pure dye from the column and a gradient of 0 to 50% acetonitrile gave a retention time value of 1.909 and very sharp A strong peak;

ix. The FT-IR signal of sulfates generated in the range of 1210-1150 and 1060-1030 and 650 means that sulfur is present in the compound in an O—SO ₂ — type bond;
x. 1090-1020 strong absorption band of silicates found in (1068) is silicon, means that present in the compound as -Si-O-Si; CH ₃ stretching signal, the 2950-2850 Seen; there is no amide signal and the compound is not a protein;
xi. The hydroxy stretching signal is strong and broad, which means a phenolic group;
xii. The compound contains a phenolic group in the phosphor portion of the compound;
xiii. The compound also includes a sterically protected quinonoid ring;
xiv. The phosphor moiety is bound to the Si-O group by the sulfated sugar moiety;
xv. Only the phenolic group of the compound produces ketoenol tautomerism, thus producing both a quinone-type structure and a phenolic structure; and xvi. The compound is used as a multiple fluorescent dye.

In another embodiment of the invention the compound has the following characteristics:
i. Not decolorized by reducing agent,
ii. The compound is not a synthetic compound,
iii. The crude extract of the pigment is yellowish green in color;
iv. The purified compound liquid pigment is brownish with green, yellow and red tones,
v. When viewed with sunlight, the purified dry compound is a reddish brown colored powder.
vi. Some green color is emitted under the tube light,
vii. Produces a bluish green under the mercury light,
viii. Under X-rays, the color is green and fluoresces,
ix. The compound is essentially amorphous;
x. The compound is water soluble and insoluble in organic solvents including ethanol, methanol and acetone;

xi. The compound is negatively charged,
xii. The compound has a pH of 2.8;
xiii. The compound comprises a quinonoid ring;
xiv. The compound is essentially non-protein-like,
xv. The compound has a reducing sugar;
xvi. The compound acts as a biosurfactant,
xvii. The compound has antibacterial properties and exhibits a zone of inhibition when an antibacterial assay is performed;
xviii. When excited with different wavelengths in the X-ray, UV and visible spectral ranges in a spectrophotometer, the compound emits fluorescence,
xix. For the compound, UV, visible spectroscopy is 250 nm-700 nm, peaks are shown at 291 nm and 451 nm wavelengths,
xx. A fluorescence spectral emission scan is performed by exciting the compound at 270 nm and a fluorescence peak occurs at 550 nm,
xvii. When the compound is excited at 340 nm, a fluorescence peak occurs at 460 nm,
xviii. When the excitation wavelength is fixed at 361 nm, the fluorescence emission reaches a maximum at 490 nm,
xix. When the excitation wavelength is fixed at 400 nm, the fluorescence emission reaches a maximum at 470 nm,
xx. When the compound is excited at 523 nm, the emission is maximum at 600 nm,

xxi. Physical examination of the dye at a concentration of 1: 2000000 dilution under a UV bulb in the 260 nm-280 nm range, ie 5 × 10 ⁻⁶ concentration, gives a bluish green fluorescence,
xxii. Fluoresce in epifluorescence microscopy of the dye at a concentration of at least 1: 200000000 fold dilution, ie 5 × 10 ⁻⁸ concentration of compounds under different fluorescent cubes,
xxiii. At four different wavelengths in the UV and visible range of the fluorescence cube of an epifluorescence microscope, the compound emits fluorescence colored in eight different colors,
xxiv. When a pure compound is excited under the excitation range of UV cubes WU-330nm-385nm, a fluorescent blue emission with indigo shades occurs in UVA in the range of 380nm-400nm,
xxv. When a pure compound is excited under a WB cube with an excitation range of 450 nm-480 nm, fluorescence green emission occurs in the range of 500 nm-570 nm,
xxvi. When a pure compound is excited under a WG cube with an excitation range of 510 nm-550 nm, a fluorescent red emission with an orange hue occurs in the range of 570 nm-650 nm,
xxvii. When viewed under a 10X, 40X, 100X objective lens, the compound emits a bluish gray tone under a normal transmitted light bulb of an epifluorescence microscope,
xxviii. Purified by epifluorescence microscopy of marine egg cells bound to fluorescent compounds at a concentration of at least 1: 200000000 dilution, ie, 5 × 10 ⁻⁸ concentration, under different fluorescent cubes. It emits fluorescence of a different color from the compound,
xxix. Cells labeled with the compounds emit fluorescence colored in four different colors at the four different wavelengths in the UV and visible range of the fluorescent cube of the epifluorescence microscope and the transmitted light bulb,

xxx. When excited in the range of 330 nm to 385 nm by a WU-fluorescence cube of an epifluorescence microscope, cells labeled with the compound fluoresce blue,
xxxi. When excited under a WB cube with an excitation range of 450 nm-480 nm, cells labeled with the compound emit bright yellow fluorescence,
xxxii. When excited under a WG cube with an excitation range of 510 nm-550 nm, the cells fluoresce red,
xxxiii. Even after one year at room temperature, the fluorescent color of the compound persists,
xxxiv. The fluorescence of the compound is highly photostable and does not degrade with long-term exposure to direct light,
xxxv. The compound does not photobleach,
xxxvi. The compound does not quench while screening slides under a fluorescence microscope; and xxxvii. When a molecule is frozen at sub zero temperature, even after freezing in liquid nitrogen, at a temperature where it is impossible to obtain the energy required for activation, as in extracts from luminescent organisms The fluorescence of the compound does not change.

  In yet another embodiment, the compound is the first natural organometallic compound that is non-radioactive and has a silicon matrix.

  In yet another embodiment, the presence of silicic acid in the compound was first identified as an essential part of a living organism.

  In yet another embodiment, the compound is a phenolic sulfated silicated polysaccharide (PSSP) and the compound is a natural for studying the relevant physiological aspects of organosilicon compounds in cosmetic surgery. An in vivo system is provided.

  In another embodiment, the compound comprises a longer wavelength, narrow bandwidth and small Stokes shift, as shown in Table 3 and Table 4, and the emission spectrum of the compound is insensitive to solvent polarity and pH. is there.

  The compound has a higher light stability, has a longer wavelength and has an emission range of one or more in the visible spectrum, and the spacer forms an essential part of the natural compound.

  In yet another embodiment, the compound has high water miscibility and facilitates easy mixing of the appropriate additives required for cosmetic and drug compositions.

  In yet another embodiment, the compound has 8 excitation (ex) / 8 emission (em), including X-ray, 270-745 nm UV and visible right spectrum with high sharp peaks. Including the nm range.

  Yet another embodiment provides a multiple fluorescent compound having excitation and emission spectra of the dye in a fluorescent filter cube in the range of ~ 450-480 nm, which is in the spectral line of an argon ion laser (~ 488 nm). Align.

  Yet another embodiment provides a multiple fluorescent dye having excitation and emission spectra of the dye in a fluorescent filter cube in the range of ~ 450-480 nm, which is an argon ion laser (~ 488 nm) such as fluorescein The cell conjugates emit a bright yellow fluorescence that forms a dye useful in multicolor applications.

  In yet another embodiment, the compound is resistant to quenching and the fluorescence intensity increases after binding to the protein, regardless of dye dilution and concentration.

  In yet another embodiment, the compound is stable at room temperature and has a long shelf life.

  In yet another embodiment, the compound molecule and radioactive kit can be exported at room temperature.

  In yet another embodiment, the fluorescence emission micrograph of the compound under fluorescence microscopy has no other special film requirements, has an exposure time in the range of 45-60 seconds, and is Kodak 400 film. It is done using.

  In yet another embodiment, the compound produces a counterstaining effect of cells and cell components under all fluorescence on a cytogenetic slide in the absence of analyte.

In yet another embodiment, even when diluted 1: 2000,000 times in ultrapure water at a concentration of 5 × 10 ⁻⁸ −10 ⁻⁶ , the compound has 8 in the X-ray, UV light and visible spectral range. When excited in one wavelength range, it emits eight colors of fluorescence.

In embodiments, the present invention relates to a novel compound having the molecular formula C ₁₀ H ₁₆ N ₂ O, which is a phosphor having the structural formula shown in FIG. 20 of the accompanying drawings.

  In yet another embodiment of the present invention, the scientific name of the phosphor is 1-hydroxy, 2,5-di-methylaminobenzene (HDAB) and has a molecular weight of 180.

Yet another embodiment of the present invention relates to a composition comprising an effective amount of a biologically active, multiple fluorescent dye compound, said compound from the marine sea cucumber sea cucumber (Hlothuria scabra) for the following useful applications: Obtained with appropriate additives.
i. Preparation of a flexible polyvinyl chloride film exhibiting a fluorescent color;
ii. The use of fluorescent colors in paints, inks, textiles;
iii. Compositions of fluorescent dyes for bleaching and whitening of polymers;
iv. Leak detection with full spectrum fluorescent dyes;
v. Use in automated chemical measurement systems;
vi. Marking the location of the destroyed shipping container; said container being selected from the group consisting of aircraft, life crafts and rockets;
vii. Submarine probe;
viii. UVA is used in photochemotherapy for skin cancer;
ix. Chromosome sunscreen component of cosmetic creams and lotions;
x. Facilitates miscibility in the humidifier due to the water miscibility properties of the dye;

xi. Fluorescent field hybridization application kit components for molecular diagnostics;
xii. Non-radioactive labeling and detection kit components, said kit being selected from the group consisting of biochemistry, cell biology, immunochemistry and molecular biology for the labeling of DNA, RNA, proteins and enzymes Used for;
xiii. Immunofluorescence detection;
xiv. Counterstaining of DIG-labeled oligonucleotide probes and anti-DIG Fab-fragments;
xv. Single and multiple flow cytometry applications;
xvi. Fluorescent staining for epifluorescence microscopy;
xvii. For rapid inspection of biological contamination in the health food industry, cosmetics industry, pharmaceutical industry, agrochemical industry and chemical industry;
xviii. For rapid assessment of biological contaminants in laboratory cultures;
xix. For rapid inspection of biocontamination factors under regional conditions;
xx. A competitive inhibitor of cholinesterase;

xxi. In an antimicrobial composition;
xxii. As a biosurfactant in toiletries compositions;
xxiii. Natural colorants;
xxiv. A bioactive composition of the dye in a ratio of 1: 20000000 in ultrapure water to obtain 8 fluorescence at 8 different wavelengths and a retardation effect under transmitted light;
xxv. Dyes for various fluorescent applications to be carried out in the subzero temperature range;
xxvi. Compounds to be used as bases for making new fluorescent probe dye derivatives;
xxvii. Compounds for molecular reagents;
xxviii. Compounds for forming conjugates for molecular applications; and xxix. To obtain phase differences and histochemical counterstaining effects for different biochemical components of cells under transmitted light.

Another embodiment of the invention relates to a method for extracting novel compounds and multiple fluorescent dyes from sea cucumber sea cucumber (Holothuria scabra) sea cucumber, said method comprising the following steps:
a. Collect animals from the shore during low tide and maintain them in laboratory, seawater containing glass tanks for taxonomic identification and further use,
b. Wash the animal thoroughly with Milli-Q water and anesthetize with chloroform / methanol in a closed bottle;
c. For lyophilization, the internal organs and skin parts of the animal are removed by peeling off with a scalpel and peeling off;
d. The freeze-dried skin of step (c) is taken up in the required amount of ultrapure water, kept on a shaker for 4 hours and then filtered on Whatman paper 1 and a sintered glass filter;
e. Evaporating the filtrate of step (d) and precipitating said compound with an organic solvent;
f. Dissolving the precipitate in a minimum volume of water and reprecipitating with an organic solvent to remove salts;
g. Centrifuge to collect residue and discard filtrate;
h. Drying the residue of step (g) to obtain a dry powder;
i. Dissolving the dry powder of step (h) in water and applying to an ion exchange column;
j. Eluting the column of step (i) with water;
k. Lyophilizing the water-eluting fraction of step (j);
l. Apply the lyophilized material of step (k) onto a DEAE cellulose column and elute with phosphate buffer;
m. Repeatedly desalting the phosphate buffer elution fraction of step (l) using a PD10 column; and n. The desired bioactive compound of the formula C ₂₂ H ₄₈ O ₆₆ N ₂ S ₁₀ Si ₇ represented by FIG. 18 or 19 is obtained.

  In an embodiment of the present invention, the marine organism Holothuria scabra is obtained from intertidal, submerged, shallow and deep waters, usually splits, fissures, ledges, swamps, overhangs, rocky and sandy habitats Abundant in shaded areas, such as the earth; insensitive to bright coloring, with or without exoskeleton and endoskeleton, plain, sticky drifters, swimming organisms, various Has the ability to swim, usually habitual and nocturnal, easy to prey on active, with or without luminescent and fluorescent dyes, all of X-ray, UVB, UVA, visible color spectrum and infrared spectrum Short light emission is generated with respect to the wavelength range.

In another embodiment of this invention, the empirical formula of the compound is C ₂₂ H ₄₈ O ₆₆ N ₂ S ₁₀ Si ₇ and has a molecular weight of 1915.

In still another embodiment of the present invention, the compound comprises a phosphor, and the phosphor has an empirical formula of C ₁₀ H ₁₆ N ₂ O and a molecular weight of 180.

  In yet another embodiment of the invention, the HPLC retention time of the multiple fluorescent dye compound was 1.909 minutes.

  In another embodiment, the compound is diluted with water at a ratio of 1: 2000,000 and the compound emits 8 fluorescence at 8 different wavelengths.

  In another embodiment, the compound is stable at different temperature ranges between subzero temperatures and 300 ° C.

In another embodiment, in FT-IR analysis, the compound exhibits a sulfate signal occurring in the range 1210-1150 and 1060-1030 and 650, which indicates that the sulfur is in an O—SO ₂ — form of the bond. Means present in the compound.

In another embodiment, FT-IR analysis confirmed that the compound exhibited a strong absorption band for silicate at 1090-1020 (at 1068) confirming the presence of -Si-O-Si- bonds. A further embodiment of the present invention provides a method of dying substances and / or components in various industrial, biological and other applications, said method comprising the required amount of claim 1 Applying or adding to the substance and / or component.

  In another embodiment, the compound is used for labeling a fluorescent molecular probe in situ hybridization (FISH).

In another embodiment, the blue colored fluorescence of the dye is comparable to the emission of the same color by the DAPI fluorescent dye at the same wavelength excitation and is a non-radioactive labeling kit for biochemistry, cell biology, immunochemistry and molecular biology Used as a component of
In another embodiment, the yellow colored fluorescence of the dye in the visible range is used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. Is comparable to
In yet another embodiment, the yellow colored fluorescence of the dye in the visible range is the same color of FITC used as a component of non-radiolabeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology Comparable to radiation.

  In yet another embodiment, the orange colored fluorescent radiation is used as a component of a non-radioactive labeling and detection kit for biochemistry, cell biology, immunochemistry and molecular biology (Propidium Iodide) Comparable to the orange fluorescent color of fluorescent dyes.

  In yet another embodiment, the orange colored fluorescent radiation is the orange fluorescence of a rhodamine fluorescent dye used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. Comparable to color.

  In another embodiment, the orange colored fluorescent radiation is the orange fluorescent color of a TRITC fluorescent dye used as a component of a non-radioactive labeling and detection kit for biochemistry, cell biology, immunochemistry and molecular biology. Comparable to

  In yet another embodiment, the dye is comparable to the color of luminescence by Hoechst 33258 used as a component in non-radiolabeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. To do.

  In yet another embodiment, the dye is based on Hoechst 33342 fluorescent dye at the same wavelength excitation used as a component of non-radiolabeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology It is comparable to the color of the light emission.

  In yet another embodiment, the compound can be used in all applications where phycobil protein is currently used, unlike the dye, where the dye does not undergo fluorescence loss upon freezing.

  In yet another embodiment, when viewed in a bright field of a fluorescence microscope under a 10X objective, the bluish gray tone produces a phase difference effect, said phase difference effect being cytogenetic, cytology Useful in rapid screening of histological and histochemical slides, the color tone saves the expense associated with components associated with the extra phase contrast of the microscope.

  In yet another embodiment, the compound, a chromatin protein of a split cell that divides into yolk, nucleoplasm and activity under a 100X oil immersion objective of a conventional transmission light microscope, is brownish in the former. Yellow, the latter yellow, and the last cell component, in a dark blue shade, show different colors of staining, which is useful in the effect of a rapid bioassay of effect, Found for chemical components.

  The present invention further provides a novel fluorescent dye, which is obtained from animal skin. The invention also describes the physical and chemical properties of the dye and its stability in direct light, especially at high and low temperatures. The dye emits eight colored fluorescences when excited at eight different wavelengths in the ultraviolet light and visible spectrum and X-ray range of fluorescence spectrophotometers and epifluorescence microscopes. The excitation and emission range of the green spectrum is the largest. The invention also relates to cell screening and cytogenetic screening for rapid examination of impurities under a fluorescence microscope. The present invention also includes advanced molecular diagnostics, epifluorescence microscopy for single and double staining of chromosomes, cells and tissues, fluorescence field hybridization applications, biosurfactants, animal therapeutics, biocontamination and leak checks, Photochemotherapy, novel remote sensing devices, underwater lobes, lifesaving devices, crushing aircraft, liferafts and defense equipment, for example, various fluorescent applications in extreme and below subzero temperatures, marking rocket positions And the use of the dyes as non-radioactive labels on protein, DNA and RNA molecular probes.

  The present invention describes fluorescent dyes obtained from marine animals, which dyes absorb sunlight or are exposed to sunlight for a longer duration due to their physiological function, at different wavelengths. Seems to have a fluorescence emission mechanism. Like phytoplankton, picoplankton and photosynthetic bacteria absorb sunlight for their photosynthetic function, use the light spectrum of the required wavelength in the chemical pathway, and emit extra light according to Stokes' law.

  Invertebrates do not have extra outer protective organs such as shells and prominent protective organs, have bioorganic chemical deposits and hard, spiny skin, and are strong internals formed from small bones and bone fragments. It has a skeleton, is sticky or slow migratory, planktonic, bottom and swimming organisms, will be exposed to direct sunlight for a long time, will live in sand or cracks and will show fluorescence.

  The present invention provides a highly effective and selective method for the extraction, purification and characterization of compounds that are fluorescent dyes from marine invertebrates, molecular diagnostics using non-radioactive labels, molecular markers, By providing components of novel instrumentation equipment for epifluorescence microscope, photochemotherapy, ground probe and subsurface probe, its diverse use for manufacturing kits for cosmetic industry, food industry etc. The aim is to overcome the disadvantages inherent in the prior art.

  The marine invertebrates are taxonomically called echinoderms called Holothuria scabra, which belongs to the steel sea cucumber class. The product of the present invention is a novel organosilicon compound, and its structure offers the possibility of fluorescence as both high and low molecular weight fluorescent dyes. The multiple fluorescent natural dye has been reported for the first time. The animals were collected from the central west coast of India during low tide, transported to the laboratory, and kept in glass tanks containing 30-32% salt water per standard. The animal was adult and sexually mature. The taxonomic position was identified as described above. In fact, most of the available dyes are synthetic in nature. There are only six types of natural pigments on the market. This includes pigments obtained from all living organisms. The fluorescent dyes reported in the present invention are the only kind from marine organisms.

  The term “organosilicon compound” as used herein is used for a Si—O—R type chemical compound in which “R” is an organic moiety. The term “phenolic and quinonoid forms of the compound” is used in the chemical structure of the phosphor portion of the compound and varies between the phenolic and quinonoid forms depending on the different electronic states and carbon bond distribution patterns. It moves and emits fluorescence and color.

  The term “dye” is used for a dye that is not decolorized by a reducing agent. The dye imparts a fluorescent color to fibers, cellulose, cell membranes and other cell components. It is called a natural pigment because the source is from marine animals commonly found in nature along the coastal, shallow or deep water world, and not a synthetic pigment. A fluorescent dye is one that, when excited at a particular wavelength, emits light within a very short duration during the transition from a higher electronic state to a lower electronic state.

  Multiple colored fluorescent dyes refer to the emission of different colored light when excited at different ranges of wavelengths. Upon excitation at different wavelength spectra of X-ray, UV and visible light, it fluoresces in indigo, blue, bluish green, green, yellowish green, yellow, orange and red tints. Cells stained with the dye emit blue, yellow and orange fluorescence. Biosurfactant means a dye solution that, if shaken, produces soapy foam and exhibits antibacterial properties. As used herein, the molecular diagnostics refer to the use of dyes as non-radioactive labels for molecular probes for fluorescent in situ hybridization applications in molecular cytogenetics and as markers in microarray and molecular biological studies. . Here, the epifluorescence microscopy is observed under different cube shapes, and when excited with a known fluorescent dye, emits a specific colored radiation, using the present dye as a dye and different colored fluorescence. It relates to microscopic studies of samples of cytogenetic slides by recording. The fluorescent dye cubes WUB, WB, WG are designated filter cube shapes with different Olympus BX-FLA reflected light fluorescence for different wavelengths.

The following steps evaluate the chemical structure and other chemical and physical characteristics of the compounded fluorescent dye:
01. Structural analysis of the fluorescent dye with compound 02. Chemical properties and empirical formula 03. Identification of the phosphor 04. Physical characteristics 05. Stability 06. Charge 07. Biosurfactant analysis,
08. FT-IR analysis,
09. Fluorescence spectrophotometry10. Physical inspection of radiation under a UV bulb in the range of 260-280 nm,
11. Preparation of cytogenetic slides by air drying method,

12 Epifluorescence microscopy of the pure dye under fluorescent dye cubes WU, WB, WG and bright field,
13. Epifluorescence microscopy of the cells under fluorescent dye cubes WU, WB, WG and bright field,
14 Staining of cytogenetic slides with the dye and screening under it,
15. Fluorescent dye cubes WU, WB, WG and bright field of BX-60 Olympus microscope,
16. 16. Fluorescence micrographs of the emitted fluorescence of pure dye under fluorescent dye cubes WU, WB, WG and bright field, and Under the fluorescent dye cubes WU, WB, WG and bright field, photomicrographs of the emitted fluorescence of the cells, and
18. Examination of the wavelength range of the fluorescent hue of the radiation and the excitation range of the fluorochrome cube,
19. Pesticide effect20. Animal therapeutics 21. Antibacterial test 22. Anti-proliferation versus biological action.

  Thus, the present invention provides natural fluorescent purified compounds and multiple fluorescent natural dyes from marine animals, which are excited at eight different ranges of wavelengths in X-ray, UV and visible light spectral wavelengths. When emitted, it emits fluorescence colored in eight different colors in the shades of indigo, blue, bluish green, green, yellowish green, yellow, orange and red.

  The present invention further relates to the emission peaks of various ranges of excitation wavelengths by recording the respective readings of the fluorescence spectrophotometer and the visible light spectrophotometer. The present invention further relates to epifluorescence microscopy of cytogenetic material on air-dried samples by using this dye as an epifluorescent microscopy dye. This dye can be used to form non-radioactive labeling kits for molecular diagnostics by fluorescent field hybridization in various molecules, biomedical and engineering sciences.

  In an embodiment, the source of the pigment is an invertebrate marine animal belonging to the subworld: metazoans, phylum: echinoderms; is there.

  In yet another embodiment, the sea cucumber (Holothuria scabra) is selected from the group consisting of sea cucumbers and is widely distributed throughout the world, particularly the Indian Ocean-West Pacific region, on the coast, shallow water, and deep water. The closest known related organisms of sea cucumber are sea urchin and starfish.

  In yet another embodiment, prior to the incision of cutaneous sea cucumber (Holothuria scabra) skin tissue, the animals were thoroughly washed with milli-Q water and anesthetized with chloroform / methanol in a closed bottle. The animals were then dissected and the skin parts were then peeled off by rubbing with a scalpel.

  In yet another embodiment, the skin was lyophilized in a commercial lyophilizer, tied at the mouth with a rubber band and stored in a plastic bag at room temperature.

  In yet another embodiment, the dye is extracted and partially purified. About 20 gm of the skin sample (lyophilized) was weighed and 200 ml of Milli-Q water was added thereto. It was then kept on a shaker and the pigment was completely extracted from the animals (4 hours).

  In yet another embodiment, the extracted pigment is then collected from the skin by decanting and then subjected to filtration using Whatman filter paper no1, then a sintered glass filter connected to a vacuum unit. did.

  In yet another embodiment, after filtration, the filtrate was collected and heated at 60 degrees Celsius on a water bath. It was transferred to a 250 ml capacity separatory funnel to which 100-150 ml of absolute alcohol was added.

  In yet another embodiment, it was also observed that methanol acetone can precipitate the dye. This is due to the change in polarity of the solvent after addition of alcohol, methanol or acetone. At that time, it was gently shaken and held for 1 hour to allow the dye to fully settle.

  In yet another embodiment, the precipitated dye was collected in a beaker by opening the separatory funnel cock.

  In yet another embodiment, it was then centrifuged at a speed of 2000 revolutions per minute using a fixed angle rotator and a glass centrifuge.

  In yet another embodiment, the precipitated dye was collected after centrifugation.

  In yet another embodiment, further purification of the dye was performed by removing sea salt by repeatedly dissolving in water and repeating the precipitation.

  In yet another embodiment, the precipitated dye was dissolved in a minimal volume of water (about 10 ml) and absolute ethanol was added thereto (50 ml). Centrifugation was performed again, and the precipitate was collected. This process was repeated 5 times in order to fully purify the dye.

  In yet another embodiment, after these steps, the dye was air dried, calibrated, and calibrated to find 2.57 gms.

  In yet another embodiment, the organic composition of the compound was found. Microanalysis of carbon, hydrogen, nitrogen and sulfur was performed from 0.25 gms of purified pigment dry powder.

  In yet another embodiment, the results of the microanalysis and atomic absorption combined with the results of the previous elemental analysis are as follows-carbon 8.572%, hydrogen 1.739%, nitrogen 0.9449% and Sulfur 9.457%, silicon dioxide 12.58% (Table 1).

  In yet another embodiment to determine the inorganic composition of the compound, the dry powder was subjected to atomic absorption photometry and silicic acid evaluation (silicon dioxide 12.58%).

  In yet another embodiment, the atomic ratio of silicon will be 7 for every 22 carbon atoms. It is noted that the results of the microanalysis and sulfur evaluation are almost the same.

  In yet another embodiment, the atomic ratio for the elements in the compound was found to be a ratio of carbon: hydrogen: nitrogen: sulfur: silicic acid :: 22: 48: 2: 10: 7.

  In another embodiment, further purification was performed by ion exchange chromatography. Cation exchange chromatography was performed using a Dowex (BioRad) column 100 sieve size. The resin was washed with 1N hydrochloric acid and immersed in the hydrochloric acid overnight. The next day it was transferred to another container and washed several times with milli-Q water. It was then poured into a 0.5-inch diameter glass column 12-inch long. The column was then washed with milli-Q water until the pH of the water eluent exiting from it was 6.5-7. In this way, the column was equilibrated with water. The aqueous solution of the compound was packed into 2.07 gms of 10 ml Milli-Q water in the column. The compound was then eluted through the column into five 25 ml fractions with milli-Q water, and for each fraction its absorbance was checked at 291 and 451, the peak to peak ratio was performed, and purity was checked. (Table 2). It can be seen from the table that the peak to peak ratios of the compounds are almost identical.

  In yet another embodiment, purification was performed by anion exchange chromatography to separate negatively charged compounds (FIG. 3a).

  In yet another embodiment, after removing the metal ions, the dye was dried by evaporation and then lyophilized.

  In yet another embodiment, silicic acid evaluation atomic absorption photometry was performed. Although metal ions do not seem to exist, the amount of silicic acid found by silicic acid evaluation was the same, 12.59%. From the above, it was confirmed that silicic acid was an incorporated part of the compound and was not present as an impurity.

  In yet another embodiment, the structure of the compound was elucidated. The compound after purification by column chromatography was subjected to acid hydrolysis. The experimental details are as follows: Approximately 50 ml of eluted sample was taken and mixed with 25 ml of 1-N hydrochloric acid. It was then attached to a reflux condenser and heated using a Bunsen burner for 1 hour. Water was continuously circulated through the reflux condenser and after 1 hour, the solution was removed in a desiccator with potassium hydroxide pellets and evaporated to dryness. The sample was removed from the desiccator after one night and added to the failing solution and the precipitate was observed. This experiment confirmed the presence of sugar units in the dye.

In yet another embodiment, the empirical formula and molecular weight were revealed. According to the atomic ratio, the formula of the compound is: C ₂₂ H ₄₈ O ₆₆ N ₂ S ₁₀ Si ₇ , molecular weight = 1915.

  In yet another embodiment, the compound is highly negatively charged and the bond between the atoms is essentially ionic.

  In yet another embodiment, the metals are not coordinated to the compound and are all ionic bonds.

  In yet another embodiment, the compound purified by column chromatography was subjected to chemical analysis for phosphor identification.

  In yet another embodiment, a pinch of zinc dust was added to 5 ml of purified fraction, to which dilute HCl (hydrochloric acid) was added, and decolorization of the solution was observed. This means the presence of a quinonoid ring.

  In yet another embodiment, mercaptoethanol was added to a purified fraction of 5 ml of compound. The compound did not cause decolorization. It can be seen that only strong reducing agents can decolorize the compound and the quinoniod ring is sterically protected, resulting in ketoenol tautomerism, resulting in both quinone-type and phenolic structures. Means only phenolic groups.

  In yet another embodiment, the purity of the compound was checked by HPLC analysis of the pure ionic form. A reversed phase C18 type analytical column with 50% acetonitrile (Isocratic) to elute the pure dye from the column produced symmetrical peaks.

In yet another embodiment, the compound was in a very diluted form (5 × 10 ⁻⁶ concentration) in an aqueous solution and visually pale yellow in color.

  In yet another embodiment, the concentrated solution was reddish brown in color, and the compounded dye was stable and did not change activity at room temperature.

  In yet another embodiment, the dye remains unchanged in fluorescence spectral properties, even after heating to 300 degrees Celsius, with no contamination or chemical decay for over a year, and its stability remains unchanged. In addition, photolysis did not proceed after exposure.

  In yet another embodiment, the marine pigments do not require stabilizers added for longer shelf life.

  In yet another embodiment, charge inspection of the dye by gel electrophoresis showed that the dye itself was a negatively charged compound that was attracted towards a positively charged electrode.

  In yet another embodiment, biosurfactant properties were assessed by shaking 0.001 mg-0.01 mg per ml solution and foaming was observed.

  In yet another embodiment, FT-IR analysis was performed. Since the compound is extremely hygroscopic, its IR is a difficult task. The compound after column chromatography was dried with a freeze dryer, mixed with potassium bromide, a pellet was prepared, and FT-IR was taken.

In yet another embodiment, in IR the sulfate signal occurring in the range 1210-1150 and 1060-1030 and 650 is seen, indicating that the sulfur is an O-SO ₂ -type bond and the compound It means that it exists inside.

  In yet another embodiment, a strong absorption band for silicate was seen between 1090-1020 (at 1068). This means that silicon is present in the compound as -Si-O-Si.

In yet another embodiment, a CH ₃ stretch signal was seen at 2950-2850.

  In yet another embodiment, the compound is not a protein because there is no amide signal.

  In yet another embodiment, the hydroxy stretching signal is also strong and broad, meaning a phenolic group.

  The inventors have studied the properties of the dyes and found that excitation at different wavelengths gives rise to multicolor emission, which is comparable to the already existing fluorochrome dyes on the market. The blue-colored fluorescence of the dye is comparable to the emission of the same color by the DAPI fluorescent dye at the same wavelength excitation and as a component of a non-radioactive labeling kit for biochemistry, cell biology, immunochemistry and molecular biology Used. The yellow colored fluorescence in the visible range of the dye is comparable to the same colored emission of auramine used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology.

  The yellow colored fluorescence of the dye in the visible range is comparable to the same colored radiation of FITC used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology.

  The orange colored fluorescent emission is comparable to the orange fluorescent color of propidium iodide fluorescent dyes used as components of non-radioactive labeling and detection kits in biochemistry, cell biology, immunochemistry and molecular biology.

  The orange colored fluorescent emission is comparable to the orange fluorescent color of TRITC fluorescent dyes used as components of non-radiolabeling and detection kits in biochemistry, cell biology, immunochemistry and molecular biology.

  The dye is stable at room temperature and has a long shelf life. The molecular kit and radioactive kit of the dye can be exported at room temperature. The dyes are at least 123 different fluorescent dyes, such as DAPI, Hoechst 33258, Hoechst 33342, FITC, acridine orange, auramine, rhodamine, TRITC and propidium iodide (they currently exist on the market ( Bitplane products))). The dye is gray under the normal light of a microscope, which produces a phase difference effect, which is useful in rapid screening of cytoplasmic, cytological and histochemical slides, This eliminates the costs associated with the extra phase difference component. The fluorescent color emission follows the Stokes law of fluorescence.

  In yet another embodiment, the fluorescence of the pure compound is seen by fluorescence spectrophotometry. The compound (freeze-dried after gel filtration) was dissolved in water (concentration 0.001 gm / ml) and subjected to fluorescence spectrophotometry. It was first subjected to excitation scanning and it was found that the compound was excited to the maximum amount at 361 nm and 523 nm wavelengths.

  In yet another embodiment, a fluorescence peak was seen at 550 nm when the compound was excited at 270 nm.

  In yet another embodiment, the fluorescence peak was 460 nm when the compound was excited at 340 nm.

  In yet another embodiment, the fluorescence peak was 490 nm when the compound was excited at 361 nm.

  In yet another embodiment, the fluorescence peak was 470 nm when the compound was excited at 400 nm.

  In yet another embodiment, the emission was maximum at 600 nm when the compound was excited at 523 nm.

In yet another embodiment, when excited with different fluorescent filter cubes for the BX60-Olympus microscope, a 5 × 10 ⁻⁸ dilution of pure compound dyed epifluorescence microscopy and light emission are recorded and the known fluorescence Compared with color tone of pigment. Pure diluted dye was added to the slide and covered with a coverslip. The screening was carried out using UV light and reflected light in the following wavelength ranges using excitation of the visible light spectrum by the Olympus WU, WB and WG cubes.

  In yet another embodiment, the wavelength range of the WU cube is 330 nm-385 nm, the wavelength range of the WB cube is 450 nm-480 nm, and the wavelength range of the WG cube is 510 nm-550 nm.

  In yet another embodiment, the emission ranges at different excitation ranges were found. Excitation in the WU 330 nm-385 nm range was seen to fluoresce in the 460 nm-490 nm range.

  In yet another embodiment, excitation with a WB filter having a spectrum in the range of 450 nm-480 nm fluoresces in the 510 nm-550 nm range.

  In yet another embodiment, excitation with a WG filter having a spectrum in the 510 nm-550 nm range fluoresces in the 610 nm-700 nm range. This result follows "Stokes' law".

In yet another embodiment, epifluorescence microscopy of an egg cell suspension in seawater of laboratory-reared marine animals is placed on a slide to which 1 μl of 5 × 10 ⁻⁸ dye is added, A cover glass was placed.

  In yet another embodiment, slide screening is performed with an epifluorescence microscope, and when excited with different fluorescent filter cubes for the BX60-Olympus microscope, the emission of light is recorded and the color tones of known fluorescent dyes Comparison. The screening is done under UV and transmitted light in the visible spectrum and fluorescent filter cubes, called WU, WB and WG cubes from Olympus.

  In yet another embodiment, the emission ranges at different excitation ranges were found. Upon excitation in the WU 330 nm-385 nm range, the cells were seen to fluoresce in the 460 nm-490 nm range.

  In yet another embodiment, upon excitation with a WB filter having a 450-480 nm range, the emission is in the spectral range of 570-610 nm.

  In yet another embodiment, upon excitation with a WG filter having a spectral range of 510 nm-550 nm, the fluorescence emission is in the range of 610 nm-700 nm.

  In yet another embodiment, epifluorescence microscopy screening of cytogenetic slides under bright areas using transmitted light radiation emitted in the full white range of the visible spectrum, depending on the density of the cell formulation components Thus, a phase difference effect was given to a bluish gray tone.

  In yet another embodiment, the fluorescent color emission of the dye under different excitation ranges was seen, the color tone emitted was noted, and the color tone was compared with known fluorescent dyes.

  Different dyes were used on different excitation cubes of the fluorescence microscope. For example, DAPI (DNA staining, emits blue), fluorescein-dUTP; Hoechst 33258, 33342 is seen under excitation with 330 nm-385 nm excitation cubes; FITC, acridine orange (for DNA, RNA, greenish / yellow Auramine under a 450 nm-480 nm excitation cube, and rhodamine, TRITC and propidium iodide under a 510 nm-550 nm excitation cube (DNA, emits an orange hue).

  In yet another embodiment, the excitation spectral range and the emission fluorescence color in epifluorescence microscopy are shown in Table 4.

  In yet another embodiment, the emitted wavelength shift observed between the purified extract of the animal, the purified compound (which is a fluorescent dye) and the cell labeled with the dye. Existed.

  In yet another embodiment, the binding of the dye to cellular components clearly demonstrated its usefulness as a fluorescent probe.

  In yet another embodiment, photomicrographs of slides with the pigment used as epifluorescence microscopy dyes were performed.

  In yet another embodiment, a photomicrograph of the emitted fluorescence within the slide with the dye under the WU 330 nm-385 nm range, WB 450 nm-480 nm range, WG 510 nm-550 nm range, and bright areas, and a 400 ASA speed Kodak Depending on the color film, 50-60 seconds of different exposures were used.

  In another embodiment, photomicrographs of the emitted fluorescence within the range of the slide with the dye under the WU 330 nm-385 nm range, with a different exposure of 50-60 seconds with a 400 ASA speed Kodak film, went. In the blue fluorescence, an indigo tone was emitted.

  In another embodiment, photomicrographs of the emitted fluorescence within the slide range with the dye under the WB 450 nm-480 nm range were performed with a 400 ASA speed Kodak film using different exposures of 50-60 seconds. It was. A green fluorescent hue was emitted.

  In another embodiment, photomicrographs of the emitted fluorescence within the slide range with the dye under the WG 510 nm-550 nm range were performed with a 400 ASA speed Kodak film using different exposures of 50-60 seconds. It was. A hue of dim red fluorescence was emitted.

  In another embodiment, under the WB 450 nm-480 nm range, the cells were subjected to photomicrographs of the emitted fluorescence within the slide range with a 400 ASA speed Kodak film using different exposures of 50 seconds-60 seconds. It was. A bright yellow fluorescent hue was emitted.

  In another embodiment, photomicrographs of emitted fluorescence within the slide range with the cells under the WG510nm-550nm range, with a 400 ASA speed Kodak film using different exposures of 50s-60s. It was. An orange fluorescent hue was emitted.

  In yet another embodiment, the agrochemical effect of the compound-attached dye was observed. The compound is toxic to insects. It was toxic to ant-like insects. The filter paper soaked in the dye was left on the work table. The next day, full of dead ants were recognized.

  In yet another embodiment, the use of cytogenetic staining dyes was made. Tissues fixed with glacial acetic acid and methanol from different sources were taken on slides and the dye solution was added thereto without pretreatment. These different parts of the cells were observed to incorporate various dye solutions. The cytoplasm, nucleus and chromosome showed different coloration. Since marine pigments stain chromosomal proteins, it adds value to studying cell karyotypes.

In yet another embodiment, the stability of the dye with compound at temperatures below zero degrees was seen. The compound in diluted form 5 × 10 ⁻⁶ was taken into a microfuge tube, maintained at −20 degrees Celsius and observed in a frozen state under UV light. The fluorescence persisted without degradation. In yet another experiment, the dye was frozen in liquid nitrogen and its fluorescence remained unchanged.

In yet another embodiment, the utility of the dye as an animal treatment for pet mites and fleas has been confirmed. The extract was used as an animal treatment to kill canine ticks / fleas. A 1: 200 dilution of 5 × 10 ⁻⁶ concentration of dye killed mites and fleas within 20 seconds.

  In yet another embodiment, this is a pigment composition for a pet mite / flea treatment, and the composition is useful as a formulation ingredient such as animal soap & cosmetics.

  In still another embodiment, an antibacterial test of the compound-attached dye was performed. Since marine pigments are phenolic compounds and phenolic compounds usually have antibacterial activity, an antibacterial assay was performed with this compound and the zone of inhibition was observed.

  In yet another embodiment, the present invention is a purified, obtained from the marine sea cucumber Hollothuria scabra to obtain 8 colors of fluorescence at 8 different wavelengths and a phase difference effect under transmitted light. Provided is a bioactive composition containing a fluorescent dye with a compound in a ratio of 1: 2000000 in ultrapure water.

  The present invention also provides a purified compound-containing fluorescent dye obtained from the marine sea cucumber Hollothuria scabra, together with conventional additives, useful for the preparation of flexible polyvinyl chloride films exhibiting a fluorescent color. A composition comprising is provided.

  In an embodiment, the present invention is a composition comprising a purified compounded fluorescent dye obtained from the marine sea cucumber sea cucumber (Holothuria scabra), together with conventional additives, useful for the preparation of coating compositions and inks. I will provide a.

  In another embodiment, the present invention provides a composition comprising a purified compounded fluorescent dye obtained from the marine sea cucumber Holothuria scabra, with conventional additives, useful for leak detection. To do.

  In yet another embodiment, the present invention provides a composition comprising a purified compounded fluorescent dye obtained from the marine sea cucumber Hollothuria scabra, with conventional additives, useful in submarine probes. To do.

  In yet another embodiment, the present invention provides a composition comprising a purified compound-attached fluorescent dye obtained from the marine sea cucumber Holothuria scabra, useful in skin cancer photochemotherapy, with conventional additives. Offer things.

  In yet another embodiment, the present invention provides a cosmetic composition comprising a purified compounded fluorescent dye, obtained from the marine sea cucumber Holothuria scabra, with conventional additives.

  In yet another embodiment, the present invention relates to a purified fluorescent dye with a compound, obtained from the marine sea cucumber sea cucumber (Holothuria scabra), useful as a fluorescent probe in a molecular diagnostic in situ hybridization kit. A composition comprising the additives of

  In yet another embodiment, the present invention is obtained from the marine sea cucumber Hollothuria scabra, useful as a component of non-radioactive labeling and biochemistry, cell biology, immunochemistry and molecular biology detection kits. Also provided is a composition comprising a purified fluorescent dye with a compound together with conventional additives.

  In yet another embodiment, the present invention provides a composition comprising a purified fluorescent dye with compound, obtained from the marine sea cucumber sea cucumber (Hlothuria scabra), with conventional additives, useful in immunofluorescence detection. I will provide a.

  In yet another embodiment, the present invention relates to a purified compound fluorescent product obtained from the marine sea cucumber Hollothuria scabra, useful as a counterstain for DIG-labeled oligonucleotide probes and anti-DIG Fab-fragments. Compositions are provided that contain the dye together with the usual additives.

  In yet another embodiment, the present invention relates to a purified fluorescent dye with a compound obtained from the marine sea cucumber sea cucumber (Holothuria scabra), useful in single and multiple cellular fluorescence in flow cytometry. Compositions comprising the usual additives are provided.

  In yet another embodiment, the present invention provides a purified compound-added fluorescent dye obtained from the marine sea cucumber Hollothuria scabra that is useful as a fluorescent stain for epifluorescence microscopy. A composition comprising the product is provided.

  In yet another embodiment, the invention is a purified, obtained from the marine sea cucumber Hollothuria scabra, useful for rapid inspection of biological contamination in the health food, cosmetics, pharmaceutical and chemical industries. Provided is a composition comprising a compounded fluorescent dye together with conventional additives.

  In yet another embodiment, the present invention provides a purified fluorescent dye with a compound, usually obtained from the marine sea cucumber Hollothuria scabra, useful for rapid assessment of biological contaminants in laboratory culture. A composition comprising the additives of

  In yet another embodiment, the present invention relates to a purified fluorescent dye with a compound obtained from the marine sea cucumber Hollothuria scabra, which is useful for the rapid examination of biofouling factors under regional conditions. Compositions comprising the usual additives are provided.

  In yet another embodiment, the present invention comprises a composition comprising a purified compounded fluorescent dye obtained from the marine sea cucumber Hollothuria scabra, together with conventional additives, useful in antibacterial compositions. provide.

  In yet another embodiment, the present invention provides a purified compounded fluorescent dye obtained from the marine sea cucumber Hollothuria scabra, useful as a biosurfactant in toiletries compositions, with conventional additives. A composition comprising is provided.

  In yet another embodiment, the present invention comprises a composition comprising a purified compounded fluorescent dye obtained from the marine sea cucumber sea cucumber (Holothuria scabra), useful as a natural colorant, with conventional additives. provide.

Explanation of Tables Table 1: Elemental analysis results of pure compounds.
Table-2: Spectrophotometer data for pure compounds.
Table-3: Fluorescence spectrophotometry of pure compounds showing excitation emission wavelengths and corresponding spectral colors.
Table-4: Said emission of different colored fluorescence of fluorescent dyes with pure compounds when excited by different wavelength fluorescent filter cubes of an Olympus epifluorescence microscope with or without conjugates.

  The present invention relates to a novel pigment extraction, partial purification and characterization process, which is widely distributed along the central west coast region of the world's India and Indo-Pacific regions. : Natural pigment derived from Holothuria scabra).

  The present invention further provides a novel fluorescent pure pigment derived from animal skin pigments, which can be repeatedly extracted 3-4 times from the same specimen by storing at -20 degrees Celsius or less, Therefore, it can be protected from exploitation of natural resources.

The present invention also contemplates that the dye has eight colored fluorescent emissions at eight different excitation wavelengths in the light spectrum (X-ray, UV and visible light spectrum). Thus, the fluorescent compounded dye is the following simple as 8 in one flurochrome dye of epifluorescence microscopy for single and double staining of chromosomes, cells and tissues: Can be commercialized according to the protocol. The dyes are presently at least 22 different fluorophores (R-phycoerythrin, Texas red, BODIPY FL, Oregon green, fluorescein, rhodamine red-X, tetramethylrhodamine, BODIPY TMR, BODIPY TR, YOYO- 1, DAPI, Indo-1, cascade blue, Fura-2, aminomethyl _{coumarin, FM 1-43, DilC 18, NBD} , carboxy -SNARF-1, Lucifer yellow, dansyl + R + NH _2, propidium iodide (Propdium iodide), etc. ) In the equivalent wavelength spectrum. Pycobili protein currently used for multicolor fluorescence detection. The dye actually covers the wavelength emission spectrum of 123 fluorescent dyes currently on the market for fluorescent probes.

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

  Thus, in a preferred mode of use, the dye is a component of a molecular labeling and detection kit, most of which are imported and sold at high prices.

  These labeling kits are widely sought for molecular diagnostics using rapid molecular cytogenetics and microarray technology.

  In yet another preferred mode of utility, fluorescence properties with X-rays can be used in some medical applications.

  This property can be used in life-saving devices to mark the location of destroyed aircraft, lifeboats and defense equipment, such as rockets, as a component of life jackets and for industrial leak checking.

  The present invention will be useful in quantitative measurements of fluorescence in flow cytometers for single and multiple cells.

  The present invention may also be advantageous in the rapid assessment of biological contamination in natural and controlled environments (eg tissue culture), contamination, industrial contamination in the health food and cosmetic industries.

  The ability of dyes to fluoresce in the UVA range when excited at low wavelengths of UV radiation is useful for selective photochemotherapy for skin cancer.

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

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

  In yet another preferred mode of use, the fluorescent dye will produce a natural color in the cosmetic and save on the cost of color additives.

  Another usefulness of the fluorescent dye is as a component of novel remote sensing devices and submarine probes that require data-based light wavelength sensitivity.

  In yet another preferred mode of utility, the dye is pre-requisite for multicolor labeling experiments (eg, DNA sequencing, flow cytometry, fluorescent in situ hybridization and fluorescence microscopy). It will be advantageous in all types of modern research.

  In yet another preferred mode of utility, the dye will be advantageous in multicolor experiments in geographical locations where the ambient temperature is below zero degrees, such as in polar and ambient locations.

  Another usefulness of the high molecular weight multiple fluorescent dye is that it is a high molecular weight dye, but the phosphor moiety is of low molecular weight and thus the dye can be useful in both options. is there.

  Another utility of the fluorescent dye is that it can be used as a base and several derivatives and conjugates can be made for industrial applications.

  The invention is illustrated by the following examples, which illustrate the scope of the invention and are not intended to be limiting in any way.

Examples Methods of extraction, purification, characterization of the pure dye with fluorescent compound, description of the chemical structure of the compound, both phenolic and quinonoid forms, the structure of the phosphor are disclosed. Details of experiments performed with a fluorescence spectrophotometer and an epifluorscence microscope to detect the emission wavelength and fluorescence color after excitation at wavelengths in the X-ray, UV and visible spectral ranges also include Disclosure.

Example: 1
Material collection Akai: metazoans Gate: Echinoderms Amon: terrestrial
Tuna: Sea cucumber
Azuna: Awate Azuna
Eye: Aspidochirota
Genus: Sea cucumber
Species: Scabra
Animals belonging to these were collected from the coast of the central west coast of India during low tide. The animals were transported to the laboratory and maintained in glass tanks containing 30-32 (30% ₀ ) salt water for each taxonomic criteria and for further use. The animal was adult and sexually mature.

Example 2:
Incision of the animal's tissue The animals were thoroughly washed with Milli-Q and anesthetized with chloroform / methanol in a bottle closed with water. The animal was then dissected and the skin portion was scraped by rubbing with a scalpel and the skin was lyophilized in a freeze dryer (HETO freeze dryer) at a cooling temperature of about -110 degrees Celsius. Centrifugal liquid concentration system).

Example: 3
Extraction and partial purification of the dye:
Approximately 20 gm of the skin sample (lyophilized) was then weighed and 200 ml of Milli-Q water was added thereto. It was then kept on a shaker and the dye was completely extracted from the animals (for 4 hours). The extracted pigment was then collected from the skin by decantation and then subjected to filtration using a Whatman filter paper no1, then a sintered glass filter connected to a vacuum unit. After filtration, the filtrate was collected and heated at 60 ° C. on a water bath. It was transferred to a 250 ml capacity separatory funnel to which 100-150 ml of absolute alcohol was added. Methanol and acetone were also observed to precipitate the dye. It is due to the change in polarity of alcohol, methanol or acetone solvents. It was then shaken gently and left for 1 hour to completely precipitate the dye. The precipitated dye was separated into a beaker by opening the separatory funnel cock. It was then centrifuged at a speed of 2000 revolutions per minute using a fixed angle rotator and a glass centrifuge. After centrifugation, the precipitated dye was collected.

Example 4:
Purification of the dye to remove sea salt:
Further purification of the dye was performed by removing sea salt by repeatedly dissolving in water and precipitating repeatedly. The precipitated dye was dissolved in a minimum volume of water (about 10 ml) and absolute ethanol was added to it (50 ml). Centrifugation was performed again to collect the precipitate. This method was repeated 5 times to sufficiently purify the dye. After performing the following steps, the dye was air dried and then weighed 2.57 gms.

Example 5:
Purification of the dye to remove counterions:
Cation exchange chromatography was performed with the aid of a Dowex (BioRad) column 100 sieve size. The resin was washed with 1N hydrochloric acid (HCl) and immersed in it overnight. The next day it was transferred to another container and washed several times with milli-Q water. It was then poured into a glass column 12-inch long and 0.5 inch diameter. The column was then washed again with milli-Q water until the pH of the water eluted therefrom was 6.5-7. In this way, the column was equilibrated with water. The compound in aqueous solution was loaded onto 2.07 gms of 10 ml Milli-Q water in the column. The compound is then eluted through the column into 5 fractions of 25 ml with milli-Q water, and for each fraction the absorbance is checked at 291 and 451 and the peak-to-peak ratio is determined to determine its purity Checked. It was found from the table that the peak-to-peak ratio of the compound remained almost the same. After removing the metal ions, the dye was dried by evaporation and then lyophilized.

Example 6:
Separation of pure compounds from partially purified dyes:
The negative constituents of the compound were separated by DEAE cellulose chromatography. A 110 ml bed volume of column was prepared with DEAE cellulose pre-swelled with phosphate buffer at pH 7.5. The column was equilibrated with phosphate buffer 0.05 (M). The lyophilized powder was dissolved in phosphate buffer and packed into the column (5-ml). A gradient of 2M NaCl was passed through the column. Fractions were collected in 5 ml aliquots. Gradient avoidance was performed up to 200 ml. After washing the column with 2M NaCl in phosphate buffer 0.05M, the fractions were collected in 5 ml aliquots. The DEAE cellulose purification peak was desalted using a desalting column (PD10) and our compound was observed to come to bed volume rather than empty volume. The compound was repeatedly desalted until there was no trace of salt present. After each run it was lyophilized (Figure 3a).

Example 7:
Bioactive peak identification:
Bioactivity and fluorescence analysis were performed on all chromatographically purified peaks. The last peak (which came out when washed with 2M NaCl) was found to be bioactive. Silicic acid was evaluated by atomic absorption analysis, and all other components were evaluated by a CHNS analyzer. According to the elemental analysis of this peak, the ratio of C: H: N: S: Si was 8.3572% carbon, 1.739% hydrogen, 9.449% nitrogen, 9.457% sulfur, and 12.58% silicon dioxide. It was shown that. When converted to elemental ratio, it is C: H: N: S: Si = 22: 48: 2: 10: 7 (Table 1).

Example 8:
Purity check of the compound:
The desalted and DEAE cellulose purified peak was dissolved in 50% acetonitrile in water and 1 mM tetra (ter) butylammonium phosphate was added as an ion-pairing reagent. It was then subjected to an analytical step using 50% acetonitrile containing 1 mM tetra (ter) butyl ammonium phosphate. A symmetrical peak was observed, thereby confirming the compound purity (FIG. 3b). A UV-visible scanning of the pure compound was taken and shown in FIG. 3c.

Example: 9
Structural description of the compound:
The compound after purification by column chromatography was subjected to acid hydrolysis. The experimental details are as follows: Approximately 50 ml of eluted sample was taken and mixed with 25 ml of 1N hydrochloric acid. Thereafter, it was attached to a reflux condenser and heated for 1 hour using a Bunsen burner to continuously circulate water through the reflux condenser. After 1 hour, the solution was removed with potassium hydroxide pellets in a desiccator and evaporated to dryness. The sample was removed from the desiccator after one night and added to the top of the failing solution and the precipitate was observed. This experiment confirmed the presence of sugar units in the dye.

Example 10:
Chemical properties and empirical formula:
According to the atomic ratio, the formula of the compound is: C ₂₂ H ₄₈ O ₆₆ N ₂ S ₁₀ Si ₇ , molecular weight = 1915. The compound was highly negatively charged and in balance it was in salt form. Metals are not bonded to the compound by coordination, and all are ionic bonds.

Example 11:
Identification of fluoropores:
The compound purified by column chromatography was subjected to chemical analysis for fluoropore identification.
(A) About 5 ml of the purified fraction was taken, and neutral ferric chloride was added thereto. A purple color was observed. This means that a phenolic group is present in the compound. A pinch of zinc dust was added to the 5 ml purified fraction, to which dilute HCl (hydrochloric acid) was added, and decolorization of the solution was observed. This means the presence of a quinonoid ring.

  Mercaptoethanol was added to 5 ml of the compound purified fraction. The compound did not decolorize. Only strong reducing agents can decolorize the compound, the quinoniod ring is sterically protected, can give ketoenol tautomerism, quinone type structure and phenolic structure It can only be a phenolic group that can produce both.

Example 12:
Physical characteristics of the compound:
The compound in pure hydrogen form has a pH of 2.8. The compound in salt form is stable up to 300 ° C. without changes in physical and chemical properties. The compound was in a very diluted form (5 × 10 ⁻⁶ concentration) in an aqueous solution, with the naked eye, the color was light yellow, and the concentrated solution was reddish brown.

Example 13:
Dye stability check:
The dye is stable and remains active at room temperature and does not change up to 300 degrees Celsius, as evidenced by no change in spectral properties after such treatment. The compound retained its stability for about a year without contamination or chemical degradation. The marine pigment did not undergo photodegradation after light treatment. Therefore, the marine pigment does not require a stabilizer.

Example 14:
Test charge of the dye by gel electrophoresis A dye sample (10 ml) was loaded onto a 1% agarose gel prepared with 0.5X TBE. The gel was run at 65 volts for 1 hour. It was removed from the gel casting system and observed under the naked eye and under a UV light transmitter. The dye was moved toward the positively charged electrode, which revealed that the dye itself was a negatively charged compound. It was therefore attracted towards the positively charged electrode.

Example 15:
Biosurfactant:
By shaking 0.001 mg-0.01 mg for each ml solution, the biosurfactant properties of the compound were evaluated and foaming was observed.

Example 16:
FT-IR analysis:
The IR spectrum has been a difficult task because the compound is inherently very hygroscopic. The compound after column chromatography was dried with a freeze dryer, mixed with potassium bromide to prepare a pellet, and then used in FT-IR analysis. In IR, the sulfate signal occurring in the range 1210-1150 and 1060-1030 and 650 is seen, which means that sulfur is present in the compound in an O-SO ₂ -type bond. To do. For silicates, a strong absorption band was seen between 1090-1020 (at 1068). This means that silicon is present in the compound as -Si-O-Si. A CH ₃ stretch signal was seen between 2950-2850. Since there is no amide signal, it is certain that it is not a protein. The hydroxy stretching signal is strong and broad, meaning a phenolic group.

Example 17:
Fluorescence properties of pure compounds by fluorescence spectrophotometry:
The compound (freeze-dried after gel filtration) was dissolved in water (concentration 0.001 gm / ml) and subjected to fluorescence spectrophotometry.

  It was first subjected to excitation scanning, confirming that the compound was excited to the maximum amount at these wavelengths of 361 nm and 523 nm.

When the compound (which is a fluorescent dye) was excited at 270, a fluorescence peak was observed at 550.
Its fluorescence peak was 460 when excitation was performed at 340 nm.
Its fluorescence peak was 490 when excitation was performed at 361 nm.
Its fluorescence peak was 470 when excitation was performed at 400 nm.
When the compound was excited at 523 nm, the emission was maximum at 600 nm.

Example 18:
Epifluorescence microscopy of pure dyes:
Record the emission of light when excited with different fluorescent filter cubes for the BX 60-Olympus microscope and color tone with known fluorescent dyes at 5 × 10 ⁻⁸ dilution and epifluorescence microscopy study Compared. Pure diluted dye was added to the slide and covered with a coverslip. Screening was performed with excitation of UV light and visible light spectrum by WU, WB and WG cubes of Olympus reflected light in the following wavelength range.
The wavelength range of the WU cube was 330 nm-385 nm.
The wavelength range of the WB cube was 450 nm to 480 nm.
The wavelength range of the WG cube was 510 nm-550 nm.

  The emission ranges at different excitation ranges were found. Excitation in the WU 330 nm-385 nm range was observed to fluoresce in the 460 nm-490 nm range. Excitation with a WB filter having a spectral range of 450 nm-480 nm emitted fluorescence in the 510 nm-550 nm range. Excitation with a WG filter having a spectral range of 510 nm-550 nm emitted fluorescence in the 610 nm-700 nm range. This result follows "Stokes' law".

Example 19:
Epifluorescence microscopy of cells:
The suspension of marine animals bred in the laboratory in seawater was placed on a slide, to which 1 μl of 5 × 10 ⁻⁸ dye was added and a cover glass was placed. The slides were screened under an epifluorescence microscope, the light emission when recorded by different fluorescent filter cubes for the BX60-Olympus microscope was recorded, and the colors were compared with known fluorescent dyes. Screening was performed under transmitted light and fluorescent filter cubes in the UV and visible light spectrum called WU, WB and WG cubes by Olympus. An emission wavelength based on the color emitted by the cells was performed.

  The emission ranges at different excitation ranges were found. With excitation having a WU 330 nm-385 nm range, the cells were confirmed to fluoresce in the 460 nm-490 nm range.

  Upon excitation with a WB filter having a 450-480 nm range, the emission was in the spectral range 570-610 nm.

  Upon excitation with a WG filter having a spectral range of 510 nm-550 nm, the fluorescence emission was in the range of 610 nm-700 nm.

  Epifluorescence microscopic screening of cytogenetic slides using transmitted light emission in the full white range of visible light and depending on the density of the cell formulation components under bright areas to a bluish gray shade. A phase-like effect was produced.

Example 20:
Fluorescent color emission of dyes under different excitation ranges:
The color of the emitted tone was recorded. The excitation spectral range and emitted fluorescence color are shown in Table 5. A shift in the emission wavelength was observed between the purified extract of the animal and the purified compound (which is a dye and cells are stained with the dye) when the dye was excited with a cell suspension. . This may be due to the binding of the dye to cell components. The usefulness as a fluorescent probe was clearly shown.

Example 21
Micrographs of slides with pigments used as epifluorescence microscopy dyes Range of slides in the WU 330nm-385nm range, WB450nm-480nm range, WG510nm-550nm range and bright areas with and without cells Photomicrographs of radiant fluorescence were taken on a 400 ASA speed Kodak color film with various exposure times of 50-60 seconds. All or part of the same thing was converted to black and white.

Example 22
Pesticide effect The compound was toxic to insects. It was toxic to ant-like insects. The filter paper soaked in the dye was left on the work table. The next day, many dead ants were found.

Example 23
Cytogenetic staining with the dye Tissues fixed in glacial acetic acid and methanol from different origins were taken on slides and the dye solution was added thereto without pretreatment. Different parts of the cells were observed to take up the dye solution differently. The cytoplasm, nucleus and chromosome showed different coloration. The marine pigments have increased value in cell karyotype studies because they stain chromosomal proteins.

Example 24
Stability of the dye with compound at temperatures below zero degrees:
The compound in diluted form 5 × 10 ⁻⁶ was placed in a microphage tube, maintained at −20 degrees Celsius, and observed in a frozen state under UV light. The fluorescence persisted without degradation. In another experiment, the dye was frozen in liquid nitrogen and the fluorescence remained unchanged.

Example 25:
Animal remedies:
The extract was used as an animal treatment for killing canine ticks / fleas. The 5 × 10 ⁻⁸ concentration of purified compound dye killed mites and fleas in less than 20 seconds.

Example 26
Antibacterial test Since marine pigments are phenolic compounds and phenolic compounds usually have antibacterial activity, an antibacterial assay was performed with this compound and a zone of inhibition was observed. E. coli (wild type) cultures were grown overnight in 50 ml McConkie broth in conical flasks (100 ml). Antimicrobial bioassay agar medium was prepared and sterilized. Thereafter, the temperature was set to 50 ° C., and 1 ml of E. coli (wild type) culture was added thereto. The culture was mixed with an antimicrobial bioassay agar medium and allowed to solidify. A 10 mg / ml sample was prepared and immersed in a filter paper disc. It was then placed on the antimicrobial bioassay agar medium inoculated with E. coli. It was then cultured at 37 ° C. for 24 hours in a bacterial incubator. A stopband was observed around the filter paper disk. This illuminated that the dye had antibacterial activity against gram-negative organisms such as E. coli.

Advantages over existing phosphor / fluorescent dye / fluorescent dye The compound is a dye, non-radioactive, a fluorescent dye derived from a natural source, not artificial.
2. The compound was the first natural organosilicon compound disclosed as having a silicon matrix, and silicic acid became an essential part of the compound. This provides a natural in vivo system for studying the relevant physiological aspects of organosilicon compounds, which are widely used in cosmetic surgery.
3. The majority of fluorescent dyes currently on the market are low molecular weight. Molecular probe INC. (Pg. 109) reports the limitations of low molecular weight fluorescent dyes.

4). Said fluorescent dyes have advantages over all these dyes. This is because it is a natural foam and has a high molecular weight.
5. The phosphor portion of the compound has a low molecular weight. Thus, the dye has the potential of both high molecular weight and low molecular weight fluorescent dyes.
6). The compounds are negatively charged and are therefore useful for molecular labeling of positively charged biopolymers.

7). The phosphor moiety has a neutral charge and thus has the potential to bind to all types of molecules.
8). The compound is permeable to cellular components.
9. HPLC of the compound for identification of the linking group is possible.

10. The emission spectrum is matched to a common optical filter used in fluorescence microscopy and does not require a special filter.
11. Unlike the BODIPY dye, the dye has two emission spectral ranges that match the spectrum of the argon laser light.
12 The dye has a longer wavelength, a narrow bandwidth and a small Stokes shift.

13. The spectrum of the dye is insensitive to solvent polarity and pH.
14 The dye is more photostable.
15. The dye has a longer wavelength and has one or more emission ranges in the visible spectrum.

16. The synthesis of the dye requires the addition of a spacer, while the present dye has a spacer in its structure.
17. The pigment is water miscible and can be an easily mixed formulation component for cosmetic and drug compositions.
18. The current single form of this dye with fluorescent compounds is comprised of 123 phosphors / fluorescent dyes currently sold on the market by Bitplane (each listed as http://www.bitplane.ch/ public / support / standard / Fluorochrome.htm) and about 96 phosphors of Molecular Probes, INC. (Richard P. Haughland, Fluorescent Probes and Research Drugs Handbook, pages 612-614) Cover the spectral range of excitation and emission.

19. The dyes all have a good quality of BODIPY FL and fluorescein-based dyes, but those shown in the fluorescent probe and research drug handbooks (14, 15, 109) by Molecular Probes, Inc. Has no drawbacks.
20. The dye has a high extinction coefficient, such as fluorescein and fluorescein-based dyes, which is a prominent green phosphor on the market.
21. The water solubility of the present dye is higher than the solubility of fluorescein.

22. The dye comprises 8 excitation (ex) / 8 emission (em) nm ranges, including X-ray, 270-745 nm UV and visible right spectrum with high and sharp peaks, Fluorescein has only an excitation and emission range of ˜494 ex / em 520 nm and its peak is broad.
23. The excitation and emission spectra of the dye in the fluorescent filter cube in the ~ 450-480nm range match the spectral line of an argon ion laser (~ 488nm), such as fluorescein, in both cases the emission color is green fluorescence This makes the dyes a competitor comparable to fluorescein, which is currently a prominent green phosphor for confocal laser scanning microscopy and flow cytometry applications.

24. The excitation and emission spectra of the dye in the fluorescent filter cube in the range of ~ 450-480nm match the spectral line of an argon ion laser (~ 488nm), like fluorescein, but the cell conjugate is multicolored Emits bright yellow fluorescence that forms a useful dye in
25. Said dyes do not exhibit photobleaching and thus have advantages over the strengths of fluorescein.
26. Unlike the fluorescent dyes currently available on the market, the dyes are non-reactive to pH. This can be beneficial while at the same time forming compositions for various applications of fluorescence.

27. The dye is resistant to quenching. The fluorescence intensity increases after binding to protein regardless of dye dilution and concentration.
28. The fluorescence emission spectrum is not broad and has a narrow spectral bandwidth but a broad range of emission spectra. The peak is sharp but will have advantages over other dyes in multicolor applications.
29. Dyes can be used for labeling molecular probes for application in fluorescent in situ hybridization (FISH).

30. Said dyes having high and low molecular weight are desirable for various applications. However, most of the currently available dyes are low molecular weight dyes. Our dye is a high molecular weight dye. Also, some of the compounds are phosphors and will be useful when low molecular weight dye substitution is required.
31. The dye is a rapid microscopic dye that provides a phase difference effect without the extra cost of phase contrast accessories of the microscope and without the long protocol of specimen fixation and storage, especially for in situ characterization of biological samples. Can be used as
32. Longer duration at room temperature, non-degradable and does not require cooling during export. The fluorescent dyes currently on the market are exported under cooling at a temperature equal to -20 degrees Celsius.

33. Longer duration, non-degradable to natural light and does not require darkness for storage.
34. Unlike the green fluorescent protein (GFP) derived from marine jellyfish, the dye with this compound is not a reporter gene. The fluorescence emission result is straightforward. The dye emits eight fluorescent colors in pure and cell-bound forms at eight different fluorescent excitation wavelengths in the X-ray, UV and visible light spectra.
35. The dyes that are highly water soluble are easily mixed in the composition where a water soluble fluorescent dye is required. The dye is insoluble in organic solvents such as ethanol, methanol and acetone.

36. The dye is negatively charged and can bind to all cell components having a positive charge.
37. The dyes have a pH of 2.8 and their spectral emission is unaffected at different pH ranges, i.e. pH 2.8-8.0.
38. The dye is essentially non-proteinaceous and non-degradable under natural conditions.

39. The dye has biosurfactant properties, thereby making it useful in soap and toiletry compositions.
40. The pigment has antibacterial properties.
41. The dyes emit these fluorescent colors even in a dilution range of 1: 2000000 times. The fluorescence of the extract is sustained at room temperature even after at least one year.

42. The polychromatic emission of the dye at different wavelengths of excitation is comparable to the fluorescent dye microscope dyes already on the market.
43. The blue-colored fluorescence of the dye is comparable to the emission of the same color by the DAPI fluorescent dye at the same wavelength excitation and is used as a component in non-radioactive labeling kits for biochemistry, cell biology, immunochemistry and molecular biology It is done.
44. The blue-colored fluorescence of the dye is comparable to the emission of color by Hoechst 33258 used as a component of non-radiolabeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology.

45. The blue-colored fluorescence of the dye is the color of the Hoechst 33342 fluorescent dye with the same wavelength excitation used as a component of non-radioactive labeling and detection kits in biochemistry, cell biology, immunochemistry and molecular biology. It is comparable to luminescence.
46. The orange colored fluorescence of the dye in the visible range is comparable to the same colored radiation of acridine orange used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology.
47. The yellow colored fluorescence of the dye in the visible range is comparable to the same colored radiation of auramin used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology.

48. The yellow colored fluorescence of the dye in the visible range is comparable to the same colored radiation of FITC used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology.
49. The orange colored fluorescent emission is comparable to the orange fluorescent color of propidium iodide fluorescent dyes used as components of non-radioactive labeling and detection kits in biochemistry, cell biology, immunochemistry and molecular biology.
50. The orange-colored fluorescent emission is comparable to the orange fluorescent color of rhodamine fluorescent dyes used as components of non-radioactive labeling and detection kits in biochemistry, cell biology, immunochemistry and molecular biology.

51. The orange colored fluorescent emission is comparable to the orange fluorescent color of TRITC fluorescent dyes used as components of non-radiolabeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology.
52. The blue-colored fluorescence of the dye is comparable to the luminescence of Hoechst 33258 used as a component in non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology.
53. The blue-colored fluorescence of the dye is due to the emission of color by Hoechst 33342 at the same wavelength excitation used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. Is comparable.

54. Unlike artificial commercial dyes used for the same purpose, the dyes are stable at room temperature and have a long shelf life. The dye molecule non-radioactive kit can be exported at room temperature.
55. The single dye has the characteristics of at least 123 different fluorescent dyes currently on the market (DAPI, Hoechst 33258, Hoechst 33342, FITC, acridine orange, auramine, rhodamine, TRITC and propidium iodide, etc.).
56. The present multiple fluorescent dyes can now be used in all applications where phycobiliproteins are used, and unlike these dyes, the dyes do not lose fluorescence upon freezing.

57. When excited in the X-ray, UV light, and visible spectral ranges of the eight wavelength ranges by the compound and multiple fluorescent dyes, even in ultrapure water (5 × 10 ⁻⁶ concentration), 1: 2000,000 times dilution, 8 Two colors of fluorescence are produced.
58. With the compound and multiple fluorescent dyes, X-ray, UV light and visible in the eight wavelength ranges, even under dilution under normal light in a microscope, ultrapure water (5 × 10 ⁻⁸ concentration), 1: 200000000 fold When excited in the spectral range, it produces eight colors of fluorescence. Gray tone produces the phase difference effect, which is useful in rapid screening of cytoplasmic, cytological and histochemical slides, and the color tone is extra for components associated with microscope phase contrast Save money. The fluorescent color emission follows the Stokes law of fluorescence.
59. The compounds and our multiple fluorescent dyes have the potential of both high and low molecular weight fluorescent dyes.

60. Fluorescence micrographs can be made with Kodak 400 film, which is now readily available on the market, and no dependence on new films is required.
61. A cytogenetic slide seen under all fluorescence produces a counterstaining effect on the cells in the background where no analyte is present but only the dye is present.
62. The said pigment | dye can be used for preparation of the polyvinyl chloride film which shows a fluorescent color. It can also be used in fluorescent colors in various paints, inks and fabrics.

63. The dye can be used in a composition of a bleaching fluorescent dye and a whitening polymer. The dye is a full spectrum fluorescent dye and can be used in leak detection. It can also be used in an automatic chemical measurement system. It can also be used to mark the location of collapsed aircraft, lifeboats and equipment, such as rockets. Furthermore, it can be used in a submarine probe. The pigment can be used in photochemotherapy for skin cancer.
64. The pigment can be used as a chromophoric sunscreen component of cosmetic creams and lotions.

65. Due to the water miscibility characteristics of the dye, miscibility in the humidifier can be facilitated. It can be used as a component of a fluorescent field hybridization application kit for molecular diagnostics. It includes DNA, RNA, proteins and enzymes, immunofluorescence detection, counterstaining of DIG-labeled oligonucleotide probes and anti-DIG Fab-fragments, single and multiple cytoquantitative fluorescence in flow cytometry, for epifluorescence microscopy It can also be used as a component of non-radioactive labeling and detection kits of biochemistry, cell biology, immunochemistry and molecular biology for the labeling of fluorescent stains.
66. The dyes can be used for rapid inspection of biological contaminants in the health food industry, cosmetics industry, pharmaceutical industry and chemical industry, for rapid assessment of biological contaminants in laboratory cultures, Can be used for proper inspection. It is also a competitive inhibitor of cholinesterase.

67. The dye can be used in an antibacterial composition. The said pigment | dye can be used as a biosurfactant in a toilet article composition.
68. The pigment is a marine pigment natural colorant A bioactive composition in a ratio of 1: 2000000 in ultrapure water to obtain 8 fluorescence at 8 different wavelengths and a retardation effect under transmitted light. Can be used.
69. At a 1: 200000000 dilution ratio with water as binder, the bioactive composition of the dye produces 8 fluorescence at 8 different wavelengths.

FIG. 1 shows a flow chart for extraction and purification of the compound / multiple fluorescent dye. FIG. 2 shows a flow chart for further purification and characterization of the compound / multiple fluorescent dye. FIG. 3 shows the separation of the compounds by DEAE cellulose chromatography. FIG. 3b is reverse phase chromatography of the pure compound, FIG. 3c is scanning of the pure compound. FIG. 4 is an IR analysis of the pure compound. FIG. 5 shows fluorescence spectrophotometry excited at 270 nm. FIG. 6 shows fluorescence spectrophotometry excited at 340 nm. FIG. 7 shows fluorescence spectrophotometry excited at 361 nm. FIG. 8 shows fluorescence spectrophotometry excited at 400 nm. FIG. 9 shows fluorescence spectrophotometry excited at 523 nm. FIG. 10 is a black and white view of an epifluorescence microscope blue fluorescent emission with a WU cube having an excitation range of 330-385 nm. FIG. 11 is a colored photograph of epifluorescence microscope blue fluorescent radiation by a WU cube with an excitation range of 330-385 nm. When the dye has no cells as a background, the color of the fluorescence has an indigo tone, while the fluorescence of cells bound to the dye is blue. FIG. 12 is a black and white view of an epifluorescence microscope green & yellow fluorescent emission with a WB cube having an excitation range of 450-480 nm. FIG. 13 is a colored photograph of epifluorescence microscope blue fluorescent radiation by a WB cube having an excitation range of 450-480 nm. When the dye is in the background and without cells, the fluorescence color is green, while the fluorescence of cells bound to the dye is yellow. FIG. 14 is a black and white view of an epifluorescence microscope red & orange fluorescent emission by a WG cube with an excitation range of 500-550 nm. FIG. 15 is a colored photograph of epifluorescence microscope red fluorescent radiation by a WG cube with an excitation range of 500-550 nm. FIG. 16 is a black and white view of an epifluorescence microscope under transmitted light cells at 100 × showing the phase difference effect. FIG. 17 shows an epifluorescence microscope under transmitted light cells at 100 × showing a bluish gray shade dye, gray shade cells and their constituents that produce counterstaining and phase contrast effects. Coloring pictures. FIG. 18 shows the chemical structure of a pure compound phenolic foam. FIG. 19 shows the chemical structure of the pure compound quinonoid form. FIG. 20 shows the structure of the phosphor.

Claims (50)

A novel multiple fluorescent dye compound having the empirical formula C ₂₂ H ₄₈ O ₆₆ N ₂ S ₁₀ Si ₇ , having a molecular weight of 1915, and having the structural formula represented by FIG. 18 or 19 of the accompanying drawings. Having the structure shown in FIG. 20, the novel compounds of claim 1 comprising a phosphor molecular formula C ₁₀ H ₁₆ N ₂ O.   The compound of claim 2, wherein the phosphor has the scientific name 1-hydroxy, 2,5-di-methylaminobenzene (HDAB), which has a molecular weight of 180. The novel compound of claim 1 having the following characteristics:
i. The organic composition of the pure compound by elemental analysis shows carbon 8.572, hydrogen 1.739%, nitrogen 0.9449% and sulfur 9.457%;
ii. The atomic ratio for the element in the compound is the carbon: hydrogen: nitrogen: sulfur ratio shown by microanalysis = 22: 48: 2: 10;
iii. Subjecting the inorganic composition of the compound to atomic absorption photometry to estimate a silicon dioxide fraction of 12.58% and silicic acid;
iv. Silicic acid is an essential part of the compound;
v. The atomic ratio of silicon is 7 for every 22 carbon atoms;
vi. Further purification of the compound by ion exchange chromatography and confirmed that the absorbance peak-to-peak ratio at 291 and 451 is stable;
vii. The compound comprises a sugar unit;
viii. HPLC of the pure ionic form of the compound with a reverse phase C18 type analytical column eluting the pure dye from the column and a gradient of 0 to 50% acetonitrile gave a retention time value of 1.909 and very sharp A strong peak;
ix. The FT-IR signal of sulfates generated in the range of 1210-1150 and 1060-1030 and 650 means that sulfur is present in the compound in an O—SO ₂ — type bond;
x. 1090-1020 strong absorption band of silicates found in (1068) is silicon, means that present in the compound as -Si-O-Si; CH ₃ stretching signal, the 2950-2850 Seen; there is no amide signal and the compound is not a protein;
xi. The hydroxy stretching signal is strong and broad, which means a phenolic group;
xii. The compound contains a phenolic group in the phosphor portion of the compound;
xiii. The compound also includes a sterically protected quinonoid ring;
xiv. The phosphor moiety is bound to the Si-O group by the sulfated sugar moiety;
xv. Only the phenolic group of the compound results in ketoenol tautomerism, thus resulting in both a quinone-type structure and a phenolic structure; and xvi. The compound is used as a multiple fluorescent dye. The compound of claim 1, wherein the compound has the following characteristics:
i. Not decolorized by reducing agent,
ii. The compound is not a synthetic compound,
iii. The crude extract of the pigment is yellowish green in color;
iv. The liquid dye of the purified compound is brownish with green, yellow and red tones,
v. When viewed with sunlight, the purified dry compound is a reddish brown colored powder.
vi. Under the tube light, some green tone is emitted,
vii. Produces a bluish green under the mercury light,
viii. Under X-rays, the color is green and fluoresces,
ix. The compound is essentially amorphous;
x. The compound is water soluble and insoluble in organic solvents including ethanol, methanol and acetone;
xi. The compound is negatively charged,
xii. The compound has a pH of 2.8;
xiii. The compound comprises a quinonoid ring;
xiv. The compound is essentially non-protein-like,
xv. The compound has a reducing sugar;
xvi. The compound acts as a biosurfactant,
xvii. The compound has antibacterial properties and exhibits a zone of inhibition when an antibacterial assay is performed;
xviii. When excited with different wavelengths in the X-ray, UV and visible spectral ranges in a spectrophotometer, the compound emits fluorescence,
xix. For the compound, UV, visible spectroscopy is 250 nm-700 nm, peaks are shown at 291 nm and 451 nm wavelengths,
xx. A fluorescence spectral emission scan is performed by exciting the compound at 270 nm and a fluorescence peak occurs at 550 nm,
xxi. When the compound is excited at 340 nm, a fluorescence peak occurs at 460 nm,
xxii. When the excitation wavelength is fixed at 361 nm, the fluorescence emission reaches a maximum at 490 nm,
xxiii. When the excitation wavelength is fixed at 400 nm, the fluorescence emission reaches a maximum at 470 nm,
xxiv. When the compound is excited at 523 nm, the emission is maximum at 600 nm,
xxv. Under a UV bulb in the 260 nm-280 nm range, a physical examination of the dye at a concentration of 1: 2000000 dilution, ie 5 × 10 ⁻⁶ concentration, gives a bluish green fluorescence,
xxvi. Fluoresce in epifluorescence microscopy of the dye at a concentration of at least 1: 200000000 fold dilution, ie 5 × 10 ⁻⁸ concentration of compounds under different fluorescent cubes,
xxvii. At four different wavelengths in the UV and visible range of the fluorescence cube of an epifluorescence microscope, the compound emits fluorescence colored in eight different colors,
xxviii. When a pure compound is excited under the excitation range of UV cubes WU-330nm-385nm, a fluorescent blue emission with indigo shades occurs in UVA in the range of 380nm-400nm,
xxix. When a pure compound is excited under a WB cube with an excitation range of 450 nm-480 nm, fluorescence green emission occurs in the range of 500 nm-570 nm,
xxx. When a pure compound is excited under a WG cube with an excitation range of 510 nm-550 nm, a fluorescent red emission with an orange hue occurs in the range of 570 nm-650 nm,
xxxi. When viewed under a 10X, 40X, 100X objective lens, the compound emits a bluish gray tone under a normal transmitted light bulb of an epifluorescence microscope,
xxxii. Purified by epifluorescence microscopy of marine egg cells bound to fluorescent compounds at a concentration of at least 1: 200000000 dilution, ie, 5 × 10 ⁻⁸ concentration, under different fluorescent cubes. It emits fluorescence of a different color from the compound,
xxxiii. Cells labeled with the compounds emit fluorescence colored in four different colors at the four different wavelengths in the UV and visible range of the fluorescent cube of the epifluorescence microscope and the transmitted light bulb,
xxxiv. When excited in the range of 330 nm to 385 nm by a WU-fluorescence cube of an epifluorescence microscope, cells labeled with the compound fluoresce blue,
xxxv. When excited under a WB cube with an excitation range of 450 nm-480 nm, cells labeled with the compound emit bright yellow fluorescence,
xxxvi. When excited under a WG cube with an excitation range of 510 nm-550 nm, the cells fluoresce red,
xxxvii. Even after one year at room temperature, the fluorescent color of the compound persists,
xxxviii. The fluorescence of the compound is highly photostable and does not degrade with long-term exposure to direct light,
xxxix. The compound does not photobleach,
xl. The compound does not quench while screening slides under a fluorescence microscope; and xli. When the molecule is frozen at subzero temperatures, even after freezing in liquid nitrogen, at a temperature where it is not possible to obtain the energy required for activation, as in extracts from luminescent organisms, The fluorescence does not change.   The compound of claim 1, wherein the compound is non-radioactive.   The compound of claim 1 which is the first natural organometallic compound having a silicon matrix.   2. A compound according to claim 1 wherein the presence of silicic acid in the compound was first identified as an essential part of a living organism.   The compound according to claim 1, wherein the compound is a phenolic sulfated silicated polysaccharide (PSSP).   The compound of claim 1, wherein the compound provides a natural in vivo system for studying the relevant physiological aspects of organosilicon compounds in cosmetic surgery.   The compound of claim 1, wherein the compound comprises a longer wavelength, a narrow bandwidth, and a small Stokes shift, as shown in Tables 3 and 4.   The compound of claim 1, wherein the emission spectrum of the compound is insensitive to solvent polarity and pH.   The compound according to claim 1, wherein the compound has higher photostability.   The compound of claim 1, wherein the compound has a longer wavelength and has one or more emission ranges in the visible spectrum.   The compound of claim 1, wherein the spacer forms an essential part of the natural compound.   The compound of claim 1, wherein the compound has high water miscibility and facilitates easy mixing of suitable additives required for cosmetic and drug compositions.   2. The compound comprises 8 excitation (ex) / 8 emission (em) nm ranges, including X-ray, 270-745 nm UV and visible right spectrum with high sharp peaks. Compound described in 1.   The compound of claim 1 which is a multi-fluorescent compound having excitation and emission spectra of the dye in a fluorescent filter cube in the range of ~ 450-480 nm, matched to the spectral line of an argon ion laser (~ 488 nm).   Multiple fluorescent dyes with excitation and emission spectra of the dye in a fluorescent filter cube in the range of ~ 450-480 nm, matched to the spectral line of an argon ion laser (~ 488 nm) such as fluorescein, but the cells The compound of claim 1, wherein the conjugate emits bright yellow fluorescence that forms a dye useful in multicolor applications.   The compound of claim 1, wherein the compound is resistant to quenching and the fluorescence intensity increases after binding to a protein regardless of dye dilution and concentration.   The compound of claim 1, wherein the compound is stable at room temperature and has a long shelf life.   The compound of claim 1, wherein the compound molecule and radioactive kit can be exported at room temperature.   A photomicrograph of the fluorescence emission of the compound under fluorescence microscopy is performed with Kodak 400 film, having no other special film requirements, having an exposure time in the range of 45-60 seconds. 1. The compound according to 1.   2. The compound of claim 1, wherein the compound produces a counterstaining effect of cells and cell components under all fluorescence on a cytogenetic slide in the absence of an analyte. Even when diluted 1: 2000000 times in ultrapure water at a concentration of 5 × 10 ⁻⁸ −10 ^−6, the compound was excited in 8 wavelength ranges of X-ray, UV light and visible spectral range. 2. A compound according to claim 1 which sometimes fluoresces in eight colors. A compound having the molecular formula C ₁₀ H ₁₆ N ₂ O, which is a phosphor having the structural formula shown in FIG. 20 of the accompanying drawings.   27. The compound of claim 26, wherein the phosphor has the scientific name 1-hydroxy, 2,5-di-methylaminobenzene (HDAB) and has a molecular weight of 180. A composition comprising an effective amount of a bioactive, multiple fluorescent dye compound according to claim 1, wherein said compound is suitable from the marine sea cucumber sea cucumber (Hlothuria scabra) for the following useful applications: Composition obtained with additives.
i. Preparation of a flexible polyvinyl chloride film exhibiting a fluorescent color;
ii. The use of fluorescent colors in paints, inks, textiles;
iii. Compositions of fluorescent dyes for bleaching and whitening of polymers;
iv. Leak detection with full spectrum fluorescent dyes;
v. Use in automated chemical measurement systems;
vi. Marking the location of the destroyed shipping container, said container being selected from the group consisting of aircraft, lifeboats and rockets;
vii. Submarine probe;
viii. UVA is used in photochemotherapy for skin cancer;
ix. Chromosome sunscreen component of cosmetic creams and lotions;
x. Facilitates miscibility in the humidifier due to the water miscibility properties of the dye;
xi. Fluorescent field hybridization application kit components for molecular diagnostics;
xii. Non-radioactive labeling and detection kit components, said kit being selected from the group consisting of biochemistry, cell biology, immunochemistry and molecular biology for the labeling of DNA, RNA, proteins and enzymes Used for;
xiii. Immunofluorescence detection;
xiv. Counterstaining of DIG-labeled oligonucleotide probes and anti-DIG Fab-fragments;
xv. Single and multiple flow cytometry applications;
xvi. Fluorescent staining for epifluorescence microscopy;
xvii. For rapid inspection of biological contamination in the health food industry, cosmetics industry, pharmaceutical industry, agrochemical industry and chemical industry;
xviii. For rapid assessment of biological contaminants in laboratory cultures;
xix. For rapid inspection of biocontamination factors under regional conditions;
xx. A competitive inhibitor of cholinesterase;
xxi. In an antimicrobial composition;
xxii. As a biosurfactant in toiletries compositions;
xxiii. Natural colorants;
xxiv. A bioactive composition of the dye in a ratio of 1: 20000000 in ultrapure water to obtain 8 fluorescence at 8 different wavelengths and a retardation effect under transmitted light;
xxv. Dyes for various fluorescent applications to be carried out in the subzero temperature range;
xxvi. Compounds to be used as bases for making new fluorescent probe dye derivatives;
xxvii. Compounds for molecular reagents;
xxviii. Compounds for forming conjugates for molecular applications; and xxix. To obtain phase differences and histochemical counterstaining effects for different biochemical components of cells under transmitted light. A method for extracting novel compounds and multiple fluorescent dyes from sea cucumber sea cucumber (Holothuria scabra) sea cucumber comprising the following steps:
a. Collect animals from the shore during low tide and maintain them in a laboratory, seawater-containing glass tank for taxonomic identification and further use;
b. Wash the animal thoroughly with Milli-Q water and anesthetize with chloroform / methanol in a closed bottle;
c. For lyophilization, the internal organs and skin parts of the animal are removed by peeling off with a scalpel and peeling off;
d. The freeze-dried skin of step (c) is taken up in the required amount of ultrapure water, kept on a shaker for 4 hours and then filtered on Whatman paper 1 and a sintered glass filter;
e. Evaporating the filtrate of step (d) and precipitating said compound with an organic solvent;
f. Dissolving the precipitate in a minimum volume of water and reprecipitating with an organic solvent to remove salts;
g. Centrifuge to collect residue and discard filtrate;
h. Drying the residue of step (g) to obtain a dry powder;
i. Dissolving the dry powder of step (h) in water and applying to an ion exchange column;
j. Eluting the column of step (i) with water;
k. Lyophilizing the water-eluting fraction of step (i);
l. Apply the lyophilized material of step (k) onto a DEAE cellulose column and elute with phosphate buffer;
m. Repeatedly desalting the phosphate buffer elution fraction of step (l) using a PD10 column, and n. The desired bioactive compound of the formula C ₂₂ H ₄₈ O ₆₆ N ₂ S ₁₀ Si ₇ represented by FIG. 18 or 19 is obtained.   The marine life sea cucumber (Holothuria scabra) is derived from intertidal, flooded, shallow and deep waters, usually shadowy, such as fissures, fissures, ledges, swamps, overhangs, rocky and sandy habitats Abundant to a certain extent; insensitive to bright coloration, with or without exoskeleton and endoskeleton, plain, sticky drifters, swimming organisms, with various swimming powers, usually , Habitual and nocturnal, easy to prey on active, with or without luminescent and fluorescent dyes, short for all wavelength ranges of X-ray, UVB, UVA, visible color spectrum and infrared spectrum 30. The method of claim 29, wherein the method produces luminescence. The method empirical formula of the compound is a _{C 22 H 48 O 66 N 2} S 10 Si 7, as claimed in claim 29 having a molecular weight 1915. Wherein said compound comprises a phosphor, the phosphor, according to claim 29 having a molecular weight of empirical formula C ₁₀ H ₁₆ N ₂ O and 180.   30. The method of claim 29, wherein the HPLC retention time was confirmed to be 1.909 minutes.   30. The method of claim 29, wherein the compound is diluted with water at a ratio of 1: 2000000, and the compound emits 8 colors of fluorescence at 8 different wavelengths.   30. The method of claim 29, wherein the compound is stable in different temperature ranges from subzero temperatures to 300 degrees Celsius. In FT-IR analysis, the compound shows a sulfate signal occurring in the range 1210-1150 and 1060-1030 and 650, which indicates that sulfur is present in the compound in an O—SO ₂ -type bond. 30. The method of claim 29, which means   The FT-IR analysis confirmed that the compound exhibited a strong absorption band for silicate at 1090-1020 (at 1068) confirming the presence of -Si-O-Si- bonds. Method.   A method of killing substances and / or components in various industrial, biological and other applications, said method comprising the required amount of the multiple fluorescent compounds of claim 1 as said substances and / or components Applying to or adding to.   39. The method of claim 38 for labeling fluorescent molecular probes in situ hybridization (FISH).   The blue colored fluorescence of the dye is comparable to the emission of the same color by the DAPI fluorescent dye at the same wavelength excitation and is used as a component of non-radioactive labeling kits for biochemistry, cell biology, immunochemistry and molecular biology 40. The method of claim 38.   39. The yellow colored fluorescence of the dye in the visible range is comparable to the same colored radiation of auramine used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. The method described in 1.   39. The yellow colored fluorescence of the dye in the visible range is comparable to the same colored radiation of FITC used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. The method described in 1.   Claims wherein the orange colored fluorescent radiation is comparable to the orange fluorescent color of propidium iodide fluorescent dyes used as components of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. 38. The method according to 38.   39. The orange colored fluorescent radiation is comparable to the orange fluorescent color of rhodamine fluorescent dyes used as components of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. The method described in 1.   39. The orange colored fluorescent radiation is comparable to the orange fluorescent color of a TRITC fluorescent dye used as a component of non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. The method described in 1.   39. The dye of claim 38, wherein the dye is comparable to the color of the luminescence by Hoechst 33258 used as a component in non-radiolabeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology. Method.   The dye is used as a component in non-radioactive labeling and detection kits for biochemistry, cell biology, immunochemistry and molecular biology, comparable to the color of the emission by Hoechst 33342 fluorescent dye at the same wavelength excitation 40. The method of claim 38.   39. The method of claim 38, wherein the compound can be used in all applications where phycobiliproteins are currently used, unlike the dye, wherein the dye does not progress in fluorescence extinction upon freezing.   When viewed in a bright field of a fluorescence microscope under a 10X objective lens, the bluish gray tone produces a phase difference effect, which is a rapid screening of cytoplasmic, cytological and histochemical slides. 40. The method of claim 38, wherein the color tone eliminates the expense associated with an extra phase difference component of the microscope. Under the 100X oil immersion objective of a conventional transmission light microscope, the compound, the chromatin protein of the dividing cell dividing into egg yolk, nucleoplasm and activity, is brownish yellow in the former, Yellow, and the last cell component, shows a different color of staining, in a dark blue shade, which is useful in the effect of a rapid bioassay of effect and is found for various histochemical components of the cell 40. The method of claim 38.

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