Drug – bio-affecting and body treating compositions – Extract – body fluid – or cellular material of undetermined... – Urogenital system
Reexamination Certificate
2002-03-28
2004-02-10
Tate, Christopher R. (Department: 1654)
Drug, bio-affecting and body treating compositions
Extract, body fluid, or cellular material of undetermined...
Urogenital system
C424S520000, C424S547000, C435S041000, C435S810000, C435S968000, C008S648000
Reexamination Certificate
active
06689391
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a bioactive extract containing a natural non-polar and non-proteinaceous fluorescent dye extracted from alcoholic extract of ovarian tissue of a marine invertebrate
Holothuria scabra
. The invention also provides a process for the extraction, partial purification and characterization of this new non-polar dye. The marine invertebrate mentioned is especially the sea cucumber. Sea cucumbers are echinoderms, members of the group of spiny skinned animals that also includes starfishes and sea urchins. Scientifically called Holothurians, they have elongated tubular bodies which are rubbery and without bony skeletons. They have the following taxonomic position.
BACKGROUND AND PRIOR ART REFERENCES
Sea cucumbers have following taxonomic position.
Subkingdom: Metazoa
Phylum: Echinodermata
Sub-Phylum: Eleutherozoa
Class: Holothuroidea
Subclasses: Aspidochirotacea, Dendrochirotacea, Apodacea
Orders: Dendrochirota, Aspidochirota, Elasipoda, Molpadonia
and Apoda
Amongst these orders sea cucumber
Holothuria scabra
belongs to:
Order: Aspidochirota
Family: Holothuriidae
Genus: Holothuria
Species:
scabra
Echinoderms are coelomate invertebrates which are exclusively marine, never colonial, are unsegmented with a basic pentameric radial, symmetry in the adult form, no head or brain, and distinguished from all other animals by structural peculiarities of skeleton and coelom. Class Holothuroidea has animals with body bilaterally symmetrical, usually elongated in the oral-aboral axis having mouth at or near one end and anus at or near the other end. The body surface is coarse, endoskeleton reduced to microscopic spicules or plates embedded in the body wall, mouth surrounded by a set of tentacles attached to water vascular system; podia or tube feet are usually present and locomotory; alimentary canal is long and coiled and cloaca usually with respiratory trees; sexes are usually separate and gonad single or paired tuft of tubules. They are sedentary forms either attached to hard substrate or burrow into soft sediments with anterior and posterior ends projected. There are more than 1000 species of holothuroids. They vary from 2 cm to 2 meter in length. They are among a few of the animals whose habitat is not restricted by ocean depths. Some of the species are reported to be making 50% of life forms at 4000 m and 90% at 8000-m depths. The species
Holothuria scabra
also called by some as
Metriatyla scabra
Jaegea is widely distributed in East Africa, Red Sea, Bay of Bengal, East India, Australia, Japan, South Pacific, Philippines, Indian Ocean and other Indo-Pacific regions. It is used for human/animal consumption in Sabah, Malaysia and Indonesia and other Indo-Pacific countries.
Fluorescent dyes are of immense value in the fluorescent probe industries to enable researchers to detect particular components of complex biomolecular assemblies, including live cells with exquisite sensitivity and selectivity (Iain D. Johnson. “Introduction to fluorescence techniques” Chapter 1.pp1 in The Handbook of Fluorescent probes and Research Chemicals by Richard P. Haughland, 6
th
edition Printed in the United States of America, 1996). Fluorescent dyes are widely used in labeling of molecular probes for localizing biological structures by fluorescence microscopy e.g in immunoassays, labeling nucleotides and oligonucleotides for in situ hybridization studies, binding to polymeric microspheres and staining of cells for use in imaging studies. Dyes are also used for selective destruction of cells such as in the technique of photodynamic therapy. (Haughland, R. P and Kang, H. C. U.S. Pat. No. 4,774,339 of Sep. 27, 1988; Haughland, R. P and Kang, H. C. U.S. Pat. No. 5,248,782, Sep. 28, 1993).
Non-polar dyes are useful in detection of neutral lipids and other non-polar liquids. The fluorophores with the non-polar quality are most suitable for making conjugants with variety of biomolecules as per the requirement of the reseachers. Two non-polar probes namely NBD probes, NBD dihexadecylamine (D-69) and NBD hexadecylamine (H-429) are primarily been used for fluorescence detection of lipids separated by TLC (The Molecular probes Handbook of Fluorescent probes and Research Chemicals by Richard P. Haughland, 6
th
edition Printed in the United States of America, 1996 pp.321-322). The same authors reported three non-polar derivatives of the bodipy fluorophore that offers an unusual combination of non-polar structure and long-wavelength absorption and fluorescence. These dyes are described to have potential applications as stains for neutral lipids and as tracers for oil and other non-polar liquids. For example BODIPY 493/503 (D-3922) is more specific for cellular lipid droplets then staining with Nile red (N-1142). BODIPY 505/515 (D-3921) rapidly permeates cell membranes of live Zebrafish embryos, selectively staining cytoplasmic yolk platelets (Mark Cooper, University of Washington quoted by Molecular probes The Handbook of Fluorescent probes and Research Chemicals by Richard P. Haughland, 6
th
edition Printed in the United States of America, 1996 pp.322). Bodipy dyes have a small fluorescence, Stokes shift extinction coeffecients that are typically greater than 80,000 cm
−1
M
−1
and high fluorescence quantum yields (0.94 for D-3921 in methanol) that are not diminished in water. Their photostability is generally high These Bodipy dyes are used in flash lamp-pumped laser dyes. Bodipy 665/676 (B-3932) can be excited using the 633 nm spectral line of the he-Ne laser or the 647 nm spectral line of the Ar-Kr laser for applications requiring long-wavelength fluorescence detection. (Richard P. Haughland, Molecular probes. The Handbook of Fluorescent probes and Research Chemicals 6
th
edition Printed in the United States of America, 1996 pp.321-322). Non-polar fluorescent dyes as dye component in industrial applications are reported in US patents (Fischer, Wolfgang; Deckers, Andreas; Guntherberg, Norbert; Jahns, Ekkehard; Haremza, Sylke; Ostertag, Werner; Schmidt, Helmut. U.S. Pat. No. 5,710,197 released on Jan. 20, 1998 entitled “Crosslinked polymer particles containing a fluorescent dye”; U.S. Pat. No. 5,304,493 dated Apr. 19, 1994 by Nowak, Anthony V Title “Method for detecting a marker dye in aged petroleum distillate fuels; U.S. Pat. No. 4,063,878 dated Dec. 20, 1977 by Weeks, Bruce W entitled “Applying sublimation indicia to pressure sensitive adhesive tape”).
At the website, http://www.uniulm.de/uni/fak
atwis/oc2/ak_wuert/publications.htm there are reported 39 publications of A. K. Wuerthner, most of which pertains to synthesis and design of chromatphores for refractive index materials and photorefractive organic glasses. About nine international patents are also disclosed (S. Beckmann, F. Effenberger, F. Würthner, F. Steybe, DE 44 16 476 (Nov. 16, 1995), WO 95/30679, Preparation of oligothiophenes for use in nonlinear optical materials; K-H. Etzbach, C. Kräh, R. Sens, F. Würthner, DE 19.611.351 (Mar. 22, 1996), WO 97/35926 (Oct. 2, 1997); Farbstoffmischungen, enthaltend Thienyl- und/oder Thiazolazofarbstoffe; Dye mixtures containing thiophene and/or thiazole azo dyes Farbstoffmischungen, enthaltend Thienyl- und/oder Thiazolazofarbstoffe; Dye mixtures containing thiophene and/or thiazole azo dyes; C. Grund, H. Reichelt, A. J. Schmidt, F. Würthner, R. Sens, S. Beckmann, DE 19648564 A1 (May 28, 1998), DE 196 50 958 A1 (Jun. 10, 1998); WO 98/23688 (Jun. 4, 1998) Indoleninmethinfarbstoffe auf Basis von Trifluormethylpyridonen; Trifluormethylpyridone Based Indolenine Methine Dyes; Indoleninmethinfarbstoffie auf Basis von Trifluormethylpyridonen; Trifluormethylpyridone Based Indolenine Methine Dyes; F. Würthner, H.-W. Schmidt, F. Haubner, DE 19643097 A1 (Apr. 23, 1998), Vernetzbare Triarylaminverbindungen und deren Verwendung als Ladungstransportmaterialien; Crosslinkable triarylamines bearing ethynyl groups for use as charge transport materials; F. Würthner, R. Sens, G. Seybold, K.-H. Etzbach, DE 197 11 445 A1 (Sep. 24, 1998), WO 98/41583 (Sep. 24, 1998)
Ganguly Anutosh
Goswami Usha
Council of Scientific & Industrial Research
Kelber Steven B.
Piper Rudnick LLP
Tate Christopher R.
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