JP2008504231A - Oxidized dibenzo-alpha-pyrone chromoprotein - Google Patents

Abstract

Composition of oxidized dibenzo-alpha-pyrone chromoprotein (DCP) and isolation of said DCP from Shilajit, ie, ammonite, coral and other invertebrate fossils. More specifically, a DCP composition comprising dibenzo-alpha-pyrone oxide or a combination thereof, phosphocreatine, protein, fatty acid acyl ester of glycerol, and other small ligands such as carotenoids, sterols and aromatic acids as core structural fragments For an explanation of things and their biological functions. Pharmaceutical, nutrition, skin care and personal care formulations are also described. These findings establish DCP as Shilajit's primary bioactive substance.

Description

REFERENCE TO RELATED APPLICATIONS This application was filed on March 12, 2004, oxygenated dibenzo - alpha - a continuation-in-part application of named pyrone dye protein U.S. Patent Application No. 10 / 799,104, the present application the entire Cited as a reference.

Background of the Invention FIELD OF THE INVENTION This invention relates to compositions of oxidized dibenzo-alpha-pyrone chromoprotein (DCP) and isolation from fossil shilajit, ammonite, coral and other invertebrates. More particularly, the invention relates to dibenzo-alpha-pyrone oxide or complexes thereof, phosphocreatine, proteins, fatty acid acyl esters of glycerol, and other small ligands as structural fragments of the core (eg carotenoids, sterols and aromatics). Acid), as well as the description of DCP compositions, including their biological functions. Pharmaceutical, nutritional, veterinary, skin care and personal care formulations are also described. These discoveries establish DCP as Shilajit's primary bioactive substance.
2. Description of Related Art There are probably thousands of carotenoproteins that can be found in nature. However, even today, the structure of only a few such compounds has been fully characterized by applying protein chemistry techniques. Partial analysis indicates that among these compounds there are many lipoproteins in which the carotenoid moiety is also associated with the lipid component. However, the stoichiometric relationship between carotenoids and proteins is not always clear.

  This application is related to US Pat. Nos. 6,440,436 B1 and 6,558,712 B1 by the same inventor, each incorporated by reference herein.

  In many chromoproteins found in living and fossil marine invertebrates, carotenoids and other colored compounds (eg, pyroloids, biliverdins and indigoids—ie, indigotin and indirubin) have been discovered and complex assemblies In addition to the association with the lipid prosthetic group, the interaction with the protein part is shown. However, there is an oxygen linker that binds to the 3- and / or 8-positions of the DBP, both in free form or in association with chromoproteins, once in a living or fossil marine invertebrate, The presence of alpha-pyrone (DBP) has never been reported. The present invention describes one such class of chromoproteins, termed dibenzo-alpha-pyrone chromoproteins (abbreviated as DCP), and has been used for Shilajit, ammonite, coral and other marine invertebrates. It is isolated in large quantities from fossils.

SUMMARY OF THE INVENTION The present invention relates to DCP compositions, isolation, and their use in treating conditions that are effective in enhancing adaptability to various stresses, such as chronic stress.

  In one embodiment, the present invention provides a dibenzo-alpha-pyrone-chromoprotein comprising dibenzo-alpha-pyrone or a derivative thereof; a phosphocreatine; a chromopeptide with a molecular weight of 2 KD or less; and a lipid having a fatty acyl ester of glycerol ( DCP) compositions are provided.

  Another embodiment of the present invention comprises dibenzo-alpha-pyrone of formula (I)

In the above formula,
R ¹ is selected from the group consisting of H, OH, O-acyl and O-amino-acyl; and R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are each independently H, Selected from the group consisting of OH, O-acyl, O-amino-acyl and fatty acid acyl groups.

  Other embodiments of the invention include compositions wherein phosphocreatine is attached to the 3 or 8 position of the dibenzo-alpha-pyrone via an ester linkage. The pigment peptide includes one or more amino acids, carotenoids and indigoids. The chromoprotein has a molecular weight of about 2 to about 20 KD.

  Other embodiments of the present invention provide skin care, hair care, pharmaceutical, veterinary or nutritional formulations comprising a DCP composition present in an amount of about 0.05 to about 50% by weight. The skin care or protection formulation can also be present in the form of a lotion, cream, gel or spray, wherein the DCP composition is present in an amount of about 0.05 to about 5% by weight.

  Other embodiments of the present invention provide said pharmaceutical formulation, wherein the pharmaceutical formulation comprises a DCP composition present in the form of a tablet, syrup, elixir or capsule.

  Another embodiment of the present invention provides the nutritional formulation comprising the DCP composition, wherein the nutritional formulation comprises about 0.5 to about 30% by weight of the DCP composition.

  Another embodiment of the present invention provides the veterinary formulation comprising the DCP composition, wherein the veterinary formulation comprises about 0.5 to about 30% by weight of the DCP composition.

  Another embodiment of the present invention provides a method of isolating a DCP composition from a Shilajit composition comprising about 0.5 to about 10% w / w dibenzo-alpha-pyrone chromoprotein, said method comprising: 1) Extracting Shilajit continuously with hot ethyl acetate and methanol and removing soluble, low or medium molecular weight organic compounds by filtration; 2) Substances insoluble in ethyl acetate and methanol, Triturating with hot water followed by pH 5.0 citrate buffer; 3) filtering the combined extraction mixture to remove insoluble materials including polymerized humic substances, minerals and metal ion salts; 4) the combined aqueous The filtrate is gradually saturated with increasing concentrations of ammonium sulfate to give a purple-brown precipitate of the DCP mixture, or the combined aqueous solution is concentrated to concentrate To precipitate a DCP such as a brownish red or off-white precipitate, filter the DCP, and evaporate the filtrate to obtain a less complex mixture of more DCP. 5) fractionating the purple-brown solid residue, which is obtained from Sephadex gel filtration and electrophoresis by ammonium sulfate saturation and isolating the DCP composition from Shilajit; Including.

  Other embodiments of the present invention provide similar methods for extracting and isolating DCP from ammonite fossils, coral fossils, and other live and post-mortem invertebrates.

Other embodiments provide a method of treating chronic stress disease, comprising administering a therapeutically effective amount of a DCP composition to a patient in need thereof, and cognitive learning comprising administering a DCP composition. Provide a way to increase.
BRIEF DESCRIPTION OF THE FIGURES FIGS . 1A and 1B show the general structure of DCP and the complex set of DCP.

  FIG. 2 shows DCP levels varying with time in red blood cells of albino rats that assimilate DCP.

  FIG. 3 shows an HPLC chromatogram of Shilajit DCP derived from a precipitate of ammonium sulfate.

  FIG. 4 shows the relationship between 3,8-dihydroxydibenzo-alpha-pyrone and the protein fraction.

Detailed Description of the Invention DCPs containing organo-mineral components exhibit orange, purple and yellow color due to oxidized carotenoids known as xanthophylls and indigoids obtained from systemic oxidation of tryptophan moieties. The Shilajit DCP exhibits a UV absorption maximum and a visible range of ˜225, ˜275, ˜320, ˜392, ˜470, ˜492, 500 535, and 620 to 660 nm in λ. The aqueous solution of DCP was diffused on silica gel having 230-400 mesh and, when heated by microwave, resulted in partial carotenoid dissociation. The identity of the colored compound was established by HPLC using a reliable marker. However, a portion of the apoprotein was obtained from this reaction and still retained most of the colored portion. Gel filtration of partially degraded proteins, and further analysis of isolated compounds (eg, chemical analysis, chromatographic analysis, and spectroscopically confirmed analysis), particularly rich in DBP and equivalents, large The existence of a prosthetic group was indicated.

Selective lipase degradation of the product liberates DBP, a phospholipid (containing C ₁₄ -C ₂₄ fatty acids, both saturated and unsaturated), and partially converts the protein into pigment lipoprotein and pigment apoprotein. Divided into. Even harsh acid hydrolysis cannot completely separate the nitrogen-containing components from the DBP-nucleus. In this way, even after classical acid hydrolysis, the lipase degradation product and some amino acids / small peptides in conjugated DBP have less polar fractions and more as determined by HPLC of the degraded product. A complex protein containing many polar fractions together still retained some of the amino acids / small peptides, xanthophylls and indigoids.

  Upon saponification, DCP produced free DBP and small conjugated DBP metabolites, namely fatty acids and amino acids. Simple removal of acylated compounds by saponification suggested that some aminoacyl and fatty acyl moieties were attached to the phenolic hydroxyl group of DBP. Furthermore, the occurrence of small O-acyl conjugation of amino acids in 3-OH-DBP from 3-O-acylglycinoyl DBP and 3-O-acyl arginoyl DBP, and creatine in DCP are shown in DCP The DBP prosthetic group structure of

In the above formula,
R ¹ = H, OH, O-acyl, O-aminoacyl, or di- or tri-peptides of these amino acids;
R ² = H or CH ₃ ;
R ³ = H or C ₁₄ -C ₂₄ saturated or unsaturated fatty acids; degree of unsaturation ranging from 1 to 6;
R ⁴ = H or C ₁₄ -C ₂₄ saturated or unsaturated fatty acids; degree of unsaturation ranging from 1 to 6; and
R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are independently selected from the group consisting of H, OH, O-acyl, O-amino-acyl and fatty acyl groups.

  The chromoprotein has a weight of 2-20 kilodaltons (KD) and includes, but is not limited to, amino acids, di- and tri-peptides of these amino acids, carotenoids and indigoids.

  Acyclic and cyclic carotenoids or xanthophylls and indigoids such as lutein, astaxanthin and beta-carotene are pigments.

  Fatty acids can be branched or unbranched, contain between 14 and 24 carbon atoms, and can be either saturated or unsaturated. The degree of unsaturation is between 1 and 6.

  Unsaturation is the number of double bonds present.

  Acyl is -COR, where R can be branched or unbranched and can contain between 16 and 18 carbon atoms, and can be either saturated or unsaturated.

  Amino acids include, but are not limited to, alanine, arginine, creatine, glycine, hydroxyproline, methionine, proline, serine, threonine and tryptophan.

  A dipeptide occurs when an amide bond is formed between one amino acid —NH 2 and a second amino acid —COOH; a tripeptide is a three amino acid bond through two amide bonds. Due to, and so on. Any number of amino acids bind together to form a long chain.

  The numbering pattern of dibenzo-alpha-pyrone is as follows.

  The presence of creatine in DCP was verified by in vivo and in vitro measurements.

  The pigment part in DCP was found to be associated with nonpolar lipids as well as the polar protein fraction. Characterisation of the degradation moiety and HPLC analysis performed after degradation of the lipase showed that the dye compound belonged to two different fractions, even in different states of binding. Upon further acid hydrolysis, the protein portion produced methionine, arginine, glycine, alanine, serine, threonine, proline and hydroxyproline as identifiable amino acids.

  DCP includes proteins with molecular weights ranging between 2KD and about 20KD. Separation of DCP into three bands by polyacrylamide gel electrophoresis (PAGE) results in a conjugate protein with a molecular weight between about 15 KD and about 20 KD present in an amount greater than about 2 KD and up to about 12 KD. However, conjugated proteins in the molecular weight range from about 12 KD to about 15 KD are present in minimal amounts.

  During elucidation of the structure of DCP, the following notable differences were noted between DCP isolated from Shilajit and DCP from Shilajit-precursor-invertebrates.

  1. DCP in which the apoprotein is colorless and colored compounds containing long prosthetic groups (eg, DBP and lipids) can be dissociated by simple treatment of an aqueous solution of DCP with either acetone or ethyl alcohol. The colorless apoprotein showed a simple HPLC pattern and generated the above-mentioned amino acids separately from DBP and its conjugation during acid hydrolysis. These DCPs are isolated from ammonite fossils and immediately separated into colorless apoproteins and colored substances, which dissolve in the extracted organic solvent.

  2. The other class constitutes DCPs where colored substances including carotenoids and indigoids cannot normally dissociate from apoproteins. This class of DCP has been isolated from Shirajit and some rare species of ammonite fossils (eg, Periphinctes with red womb).

  Some invertebrate proteins spread at the air / water interface with extreme reluctance. The apoprotein spread smoothly during electrophoresis when dissociated from a prosthetic group (eg, containing a colored substance such as a carotenoid). Carotenoids in such chromoproteins appear to act as “locks” on the tertiary or quaternary structure of the protein against denaturation. The colorless apoprotein is formed from the dissociation of chromoproteins, but in contrast undergoes rapid clotting and partial denaturation.

  In Shilajit-DCP, the association of dye molecules and apoproteins usually cannot be dissociated. There can be a specific and strong combination of the two parts. Consistent with this prerequisite, the pigment compound in Shilajit-DCP was found to be associated with both the lipid and apoprotein fractions. HPLC performed after selective degradation of DCP with lipase established this point. The stable quaternary structure of Shilajit-DCP was further suggested by the following experiment. When electrophoresed in a starch-urea gel, two chromoproteins, orange-pink (Mw ≦ 5 KD) DCP−, and yellowish-brown (DCP−) are present. A large amount of DBP was suitably included; Mw ≦ 14 KD) DCP− was isolated. These properties are that some colored (dye) molecules are covalently bound to some parts of the apoprotein and lipoprotein components. The close association between the amino acid moieties allows interaction with carotenoids and indigoids and may give the strength of the association, which is a deep deep color shift (from λ500 nm to λ660 nm). , And in fact the dark color effect in the visible spectrum of the DCP colored chromophore.

  Based on the above, the general structure of DCP (FIG. 1A) and the conjugate set of DCP (FIG. 1B) were revealed.

  The protein content of DCP was measured by the Lowry method and was 57.13%; however, it was 59.3% according to the Bradford method. The higher proportion of protein was measured by the latter method, possibly due to the higher sensitivity to a significant amount of arginine in the DCP.

A portion of the lipid moiety present in Formula 1 is covalently bonded to the prosthetic group. This was suggested by the following study. Complete extraction of DCP with a Bligh-Dyer (Bright and Dyer) solvent system was suitable for lipid extraction and gave a small amount of acylated DCP without producing any free fatty acids. The main insoluble residues upon reaction with lipase are C ₁₄ to C ₂₄ , C _{16: 0} , C _{18: 0} , and C _{18: 1} are the major components as shown in Table 1. Produced fatty acids. Thus, lipoproteins appear to constitute an integral part of the DCP.

  Many of the Shilajit-bearing mountains are rich in marine invertebrate fossils, such as the Phanerozoic Arthropoda, Brachipoda and Mollusca. It turned out to be a treasure house. This co-occurrence of Shilajit and invertebrate fossils is a consistent phenomenon, as shown in Table 2.

  Also, the organic compounds found in these fossils and shilajit are very similar to those shown in Tables 3-6.

  These findings suggest that marine invertebrates contribute to Shilajit formation.

  The marine invertebrates (Table 2) were investigated followed by DCP isolation and characterization in Shilajit. Very similar DBP-carotenoproteins and other low Mw organic and colored components (eg, indigoids) were found in the marine invertebrate samples (Tables 3-6).

  The IR spectra of a mixture of DCPs isolated from Shilajit and ammonite (Table 2) were very similar. The HPLC retention times for the main peaks and their PDA spectra were also similar. The usual work done after the DCP fraction on the complete organic solvent extract yielded astaxanthin, astaxanthin fatty acyl derivatives and canaxanthin. Amino acids isolated from 3,8-dihydroxydibenzo-alpha-pyrone and Shilajit-DCP were also isolated from the ammonite fossils (Table 2) by their acid hydrolysis.

  The colored components of the DBP-chromoprotein from the ammonite include mono-N-benzoyl indigotin, indirubin and isatin, possibly resulting from metabolism of the tryptophan moiety present in the DCP. Protein browning due to protein saccharification due to oxidative stress was also recognized in the DCP of both Shilajit and the ammonite fossil.

The maintenance of color patterns for invertebrate fossils is a rare phenomenon, but has been documented throughout the Phanerozoic. The colored molecules, including saccharification of protein products by carotenoids, indigoids, and Maillard reactions, can form stable complexes by coordination with metal ions. Such internal crystalline biomolecules act as nucleation for biomineralization. If the limb muscles of a dead marine animal are rotted, the empty space is filled with minerals, such as pyrite (FeS ₂ , CaSiO ₃ ), and then the time for the thin organic cuticles surrounding them to break down and rot. Have. The organic material forms a substrate for pyrite (and other minerals) nucleation and is ubiquitous in marine sediments. As a result of the diffusion of Fe and S into the cell, precipitation occurs. Pyrite does not directly replace the tissue, but precipitates on the surface and in space. Not only the ammonite fossils, but also the protein mutual stabilization in the colored molecules and shilajit was increased by the involvement of DBP formula (I). This is the first demonstration of the natural appearance of DBP in a complex association with chromoproteins. It was also assessed whether this association was a common phenomenon in living humans and animals. A mixture of DCPs isolated from albino rat plasma (colored pink) when compared to the corresponding fraction of DCP from Shilajit has several special features in terms of HPLC peaks and their PDA spectra. The similarity that should be shown. Even better similarity was found between semi-purified DCP components by gel filtration using Sephadex G-50 and was obtained from Shilajit and volunteer human plasma. A similar general HPLC pattern was seen in some healthy human subjects.

DCP showed active turnover for some of the key components (Figure 2) when administered to experimental animals. Similarly, when DBP was orally administered (po) to rats, it was immediately absorbed and utilized in the synthesis of DCP and related formulations. Dibenzo-alpha-pyrone oxide (DBP) is synthesized from EPA in animal ecosystems in several DBP-formulations (HPLC-t _R : 2.31, 2.99, 3.46 and 3.46). 86 minutes). These components were also detected in DCP and isolated from Shilajit. Active turnover of these components was seen with oral administration of DCP (200 mg / Kg bw) to albino rats (FIG. 2) followed by HPLC analysis of the components in the corresponding RBC. . From this and other observations, it is very clear that DCP is also a component of animal tissue and acts in the form of enzymes and hormones in regulating and realizing some biological functions. .

  DCPs are involved in producer organisms that contain protective colors that provide protection from radiation, electron transport, and enzyme activity, and their diverse functions in nutrition and development. DCP has transport properties similar to those of Shilajit fulvic acid (FAs) and can enter recipient cells and elicit much more significant biological responses than free DBP. Extensive pharmacological and immunological evaluation of DCP demonstrates that DCP is 2 to 5 times more potential than any other component such as adaptation enhancers and immunostimulants.

  Systematic conversion of 3-hydroxy- and 3,8-dihydroxydibenzo-alpha-pyrone (DBP) to arginine and glycine phospholipid complexes, their resultant metabolism, and systematic DCP The anabolic / turnover suggests a role for these compounds in the storage of energy in ecosystems when fed to rats through the oral route. Arginine phosphate plays an important role in energy storage in invertebrates; the same role is played by creatine produced from the combination of arginine phosphate and glycine phosphate in invertebrates. Creatine phosphate and arginine phosphate are phosphate reserves with high energy potential, but the name “phosphagens” is given to these compounds as shown in Scheme 1.

  When ATP is present in excess, energy coupling indicates the energy storage reaction, and conversely, when the cell requires ATP, the reverse reaction indicates formation of ATP. If the biosynthesis and balance of the DBP-phosphagen complex in living organisms is taken as an index of its energy state, in the case of deficiency of these phosphagens, administration of Shilajit (po) will replace them. It is possible.

  The chromoprotein (DCP) is involved in various functions in animal ecosystems. DCP is encountered in the lowest forms of animals (foraminifera in other marine invertebrates and termite hemolymphs), higher animals (rodents, beavers, chimpanzees, sheep), and humans.

  DCP is involved in electron transport [systematic ATP synthesis by DCP can be imagined because oral administration of DBP produced creatine and conjugate products] and other enzyme activities (eg, 1967) In Jurassic female invertebrate fossils (eg, Idiocyclocerus and Kamptokephalites spp.) (Table 2) compared to males; Large amounts of DCP have been discovered in current studies, suggesting their role in embryo development and protection. Compared to the case of EPA, DHA and free DBP formed from EPA / DHA, the superior (qualitative and quantitative) biological functions of DCP are subsequently described.

  Thus, the characteristics of DCP isolation and use are as follows.

  1. Stabilization of proteins and the colored molecules, ie carotenoids (eg astaxanthin and derivatives) and indigoids (eg indigotin and indirubin) against various forms of stress and attack.

  2. Protective colors, ie the use of colors as a means of concealment from predator predator functions; the use of highly antioxidant pigments from the harmful effects of radiation; for example, lipid photooxidation and oxidized free radicals.

  3. Development of producer organisms. The large amount of chromoproteins found in invertebrate ovaries and the higher amounts of these compounds in female species compared to males suggest their function in species development. Lipoprotein complexes that have been documented in the blood / hemolymph of many invertebrates can be involved in the transport of the carotenoids and other pigment molecules; binding to proteins that render fat-soluble pigments water soluble. However, the dye molecules in DCP were found to associate with lipids as well as the protein fraction of the complex molecule.

  4). Embryonic development in invertebrates requires carotenoprotein.

  5. As a bioenergetics simulator / alternative, eg ATP; creatine synthesis.

  6). Immune modulator.

  7. Oxidized free radical captivators, reactive oxygen species (ROS), reactive nitrogen species (RNS).

  8). Scavenger / chelator of loose metal ions (Fe, Cu, Mn, W).

  9. DCP plays an important vitalizer role in all living organisms since the evolution of life on Earth.

  The isolation and use features of DCP provide a skin care, hair care, pharmaceutical, or nutritional formulation comprising a DCP composition present in an amount of about 0.05 to about 50% by weight. The skin care or protective formulation can also be present in the form of a lotion, cream, gel or spray, wherein the DCP composition is present in an amount of about 0.05 to about 5% by weight. .

  A feature of the present invention provides a pharmaceutical formulation comprising a DCP composition, wherein the pharmaceutical formulation is present in the form of a tablet, syrup, elixir or capsule.

  A feature of the present invention provides a nutritional formulation comprising a DCP composition, wherein the nutritional formulation comprises from about 0.5 to about 30% by weight of the DCP composition.

  A feature of the present invention provides a veterinary formulation comprising a DCP composition, wherein the veterinary formulation comprises from about 0.5 to about 30% by weight of the DCP composition.

  A feature of the present invention provides a method for isolating a DCP composition from a Shilajit composition comprising about 0.5 to about 10% w / w dibenzo-alpha-pyrone chromoprotein, said method comprising: 1) Shilajit A continuous extraction with hot ethyl acetate and methanol to remove soluble low and medium molecular weight organic compounds by filtration; 2) a substance insoluble in ethyl acetate and methanol is heated to hot water followed by pH 5. Grinding with 0 citrate buffer; 3) filtering the combined extraction mixture to remove insoluble material including polymerized humic substances, minerals and metal ion salts; and 4) combining the combined aqueous filtrates. Saturate gradually with increasing concentrations of ammonium sulfate to obtain a purple-brown precipitate of a mixture of DCPs or concentrate the combined aqueous solution and add acetone to add D Precipitating P as a brownish red or off-white precipitate, filtering the DCP and evaporating the filtrate to obtain a more complex mixture of less complex DCP; 5) Sepha Fractionating the purple-brown solid residue resulting from ammonium sulfate saturation by dex gel filtration and electrophoresis, and isolating the DCP composition from Shilajit.

  Features of the present invention provide similar methods for extracting and isolating DCP from ammonite fossil, coral fossil, and invertebrates.

  The features include a method of treating a chronic stress disorder that includes administering a therapeutically effective amount of a DCP composition to a patient in need thereof, and a method of increasing cognitive learning comprising administering a DCP composition. provide.

  The following examples are provided to further characterize the nature of the invention.

Example 1
Extraction and isolation of Shilajit DCP Shilajit (rock powder) was continuously extracted with hot ethyl acetate and methanol to subsequently remove the comprehensively analyzed free organic compounds (Tables 3-6). The mark (material insoluble in ethyl acetate and methanol) was ground with hot water and citrate buffer (pH 5.0) and then filtered. The mark was analyzed for inorganic minerals and humic substances. The aqueous solutions were each saturated differently with ammonium sulfate (25%, 50%, 75% and 100%) when DCP with different complexity precipitated as a purple brown solid. The solid residue was subjected to Sephadex gel filtration and electrophoresis for further purification of DCP. A similar general procedure was performed for the purpose of isolating DCP from marine samples. However, in the precipitation of DCP from aqueous solutions, one variation constituted the addition of acetone to isolate DCP from the acetone insoluble and soluble fractions in the conventional method instead of ammonium sulfate.

Example 2
Extraction and isolation of marine invertebrate fossil DCP (general means)
In a typical experiment, a Nummulite fossil (formaminifera, GSI type 10772) was dried, finely powdered, and then extracted with hot ethyl acetate to obtain low Mw organic compounds (free dibenzo-alpha-pyrone, hydroxy) Cf. Tables 3 to 6) such as acetophenone and aromatic acid were removed as the ethyl acetate soluble fraction. The mark (insoluble in ethyl acetate) was further extracted with 0.1N HCl. The aqueous acid extract was concentrated by evaporation. The residue was dissolved with a minimum amount of distilled water. The aqueous solution was divided into two parts. One part was saturated separately from ammonium sulfate, and in the other part, acetone was gradually added. The addition of both ammonium sulfate and acetone precipitated a mixture of dibenzo-alpha-pyrone chromoprotein (DCP) oxide as a light brown solid. The acetone soluble fraction upon evaporative concentration also allowed for further recovery of less complex DCP. These compounds were subsequently subjected to chromatographic (HPLC) and spectral (IR, ¹ H-NMR) analysis to establish general identity with DCP.

Example 3
Extraction of living marine invertebrates (general means)
Surviving marine invertebrates, mainly mollusks (Telescopium, Cerethedia, etc.) were collected from the coastal waters of the Bay of Bengal and transported to the laboratory as living specimens. Each specimen was sacrificed and body flesh was removed from the shellfish. The flesh was then extracted with hot ethyl acetate to remove low molecular weight organic compounds and lipids. The mark (portion insoluble in EtOAc) is further linked to the Blye-Dyer solvent system [CHCl ₃ : MeOH (1: 2) as the initial solvent; CHCl ₃ : MeOH: H ₂ O (1: 2: as the intermediate solvent). 0.8), extracted with CHCl ₃ : MeOH (1: 2)] as the final solvent. The Briyer-Dyer (B & D) solvent was evaporated and concentrated under reduced pressure. The B & D extract was dissolved in a minimum amount of distilled water. The aqueous solution was divided into two parts. One portion was gradually saturated with ammonium sulfate and the other was gradually added with acetone. Addition of both ammonium sulfate and acetone precipitated a mixture of DCP (dibenzo-alpha-pyrone chromoprotein oxide) as an off-white solid. These compounds were analyzed by different chromatographic (HPLC) and spectral (IR, ¹ H-NMR, GC-MS) techniques to verify their identity with Shilajit DCP.

Example 4
Separation and partial characterization of DCP (and) Polyacrylamide gel electrophoresis of sodium dodecyl sulfate using 10% acrylamide in the presence of 0.1% (w / v) SDS by the method of Weber and Osborn (1969). Was executed. The sample was preheated for 3 minutes at 100 ° C. in the presence of 2-mercaptoethanol and 3% SDS. Tris-glycine buffer containing 0.1% SDS (pH 8.4) was used as the running buffer. Bromophenol blue was used as a tracking dye. Electrophoresis was performed for 90 minutes at 120V, 40 mA constant current. A pinkish orange band appeared towards the top (DCP-) followed by bromophenol blue and then towards the yellow band (DCP-). At the end of the run, these three bands were cut with a sharp blade and homogenized separately in 1.5 ml distilled water with a mortar and pestle. Each homogenate was decanted in a microcentrifuge tube and centrifuged at 7000 rpm for 10 minutes. Each supernatant was divided into two parts and evaporated under vacuum. One part of the sample (ca. 50 μg) was dissolved in HPLC running solvent (water: acetonitrile: orthophosphoric acid = 67: 32: 1) and analyzed by HPLC. The other part underwent a lipase reaction (see Example 5).

  The DCP-compound was obtained as a pink colored powder; pH (1% aqueous solution) 8.02; N, 17.8%; Metal ion (in ppm) Fe, 186.3; Cu, 8 .8; Zn, 23.4.

  The DCP-compound is obtained as a light brown powder, pH (1% aqueous solution) 7.8; N, 16.4%; metal ions (in ppm) Fe, 262.4; Zn, 48.7.

  Further purification of the two chromoproteins was performed by ion exchange of Sephadex with DEAE-Sephadex G-50 using phosphate buffer (pH 7.2). Gel electrophoresis (10% SDS, thickness 1.5 cm; constant current 20 mA, Tris-glycine buffer, pH 8.3) showed two major bands at DCP-, 2-5 KD and DCP-, 10-14 KD, respectively. With some lighter bands in the higher Mw range.

Both DCP- and DCP- showed the spectral (IR, ¹ H-NMR) characteristics typical of HPLC and DBP-carotenoprotein.

Example 5
Lipase reaction of DCP The sample (ca. 50 μg) was dissolved in 0.5 ml of 1M Tris buffer at pH 8.0. 100 μl CaCl ₂ .2H ₂ O (2.2%) and 250 μl bile salt (1%) were added to each sample. A working solution of lipase (Sigma porcine pancreatic lipase 1 mg in 2 ml Tris buffer) was then added to each sample. The mixture was stirred at 37 ° C. for 3 hours with a magnetic stirrer. After incubation, 1 ml ethanol and 1 ml 6N HCl were added to the mixture to stop the reaction. The hydrolyzed product was extracted with diethyl ether and dried over anhydrous sodium sulfate. The remaining portion was concentrated on a water bath in a porcelain basin. The residue was dissolved in a minimal amount of HPLC solvent (water: acetonitrile: orthophosphoric acid = 67: 32: 1) and 20 μl was injected into the HPLC for analysis. The recovered ether extract was also analyzed by HPLC in the same solvent system to characterize the nature of the lipid compound after lipase hydrolysis.

In the HPLC chromatogram of DCP, a number of peaks were precipitated from the aqueous solution of Shilajit by separately saturating with ammonium sulfate and appeared in 75 and 100% saturated fractions (FIG. 3). This observation suggested that Shilajit DCP was full of relatively low Mw lipoproteins (such as chylomicron / lipocalin). However, the t _R 1.5 min signal (FIG. 3) suggested that higher Mw proteins such as B-48 could also be generated in DCP. The presence of adhesive ligands, especially DBP, was also suggested.

  Another observation was the association of DBP as a ligand in DCP (FIG. 4). In this figure, PR-25, -50, -75, and -100 each mean a protein fraction in which ammonium sulfate is precipitated. In PR-50 and -75, a large amount of 3,8-dihydroxydibenzo-alpha-pyrone very well suggests that DBP is selectively associated with low / medium MW lipoproteins. Please note that.

Example 6
Determination of amino acids The mixture of amino acids produced in the acid hydrolysis of DCP is converted to trimethylsilyl derivatives (O- / N-TMS) and then subjected to GC-MS analysis by using the corresponding markers and the standard amino acids And prepared in the same manner.

Example 7
Determination of Creatine This method was described by Barrett (1936) based on the color response developed by creatine in the presence of diacetyl and alpha-naptol. Briefly, for a neutral solution of a test sample containing 60 μg or less of creatine, 2 ml of 1% alpha-naptol in alkali followed by 1 ml of diacetyl (1% solution diluted 1:20 before use) ) Was added. The solution was stirred and its color was measured after 30 minutes at 525 mμ.

Example 8
Determination of Arginine Arginine was isolated from DCP by selective degradation (lipase), degraded to ornithine and urea by arginase (5-10 units / ml) and assayed colorimetrically (acid mixture, ie 1vol of _H 2 _SO 4; 3 vol syrup _H 3 _PO 4; 1vol of _{H 2} O; _H 2 O urea 50 [mu] g / ml in Standard; and alpha in 95% ethyl alcohol 100 ml - isonitrosopropiophenone non 4g , Use).

Example 9
Comparative study on the effects of Shilajit components on chronic stress A comparative study on the bioactive components of Shilajit from EPA, DHA, DBP and DCP was conducted to determine the efficacy of albino to enhance its adaptability to chronic stress (CS) in rats did. A warm but unpredictable natural CS that cannot be confronted by animals (stress unavoidable) is medically more relevant than acute stress, even when acute stress is severe in nature, Now it is becoming more and more evidence. Chronic, unpredictable and unavoidable stress is believed to ultimately resemble the situation faced by individuals resulting in physiological worries and illnesses induced by chronic stress.

animal. The study was performed in CF-strained albino rats (140-180 g) living in a colony cage with a cycle of one sex and atmospheric temperature of 25 ± 2 ° C., 12 hours bright and 12 hours dark. The experiment was performed between 0900 hours and 1400 hours.
A 1: 1 mixture of EPA, eicosapentaenoic acid DHA, docosahexaenoic acid DBP, 3-hydroxy- and 3,8-dihydroxydibenzo-alpha-pyrone DCP, DBP-chromoprotein Induction of chronic stress Armario et al (1993) Followed. In short, rats were randomly assigned to a control or stress group. Rats assigned to the stress group received a 1 hour foot shock through the grid floor daily for 14 days. The duration of each shock (2 mA) and the interval between the shocks were each randomly programmed between 3-5 seconds and 10-110 seconds, making the stress unpredictable. The shock chamber had high walls that made it impossible to escape from the shock.

Test compounds and vehicle EPA (Aldrich, Milw.), DHA (Sigma), DBP and DCP are separately suspended / dissolved in 0.3% carboxymethylcellulose (CMC) in distilled water and administered orally for 14 days. (Po) On the first day (day 1), an electric shock was performed 60 minutes after the start. Control animals have a p.c. of 2.5 ml / kg. o. In the same period, rats received only vehicle in rats with or without stress. Evaluation was performed on day 14 (day 14) for 1 hour after the final stress procedure and 2 hours after the final test compound or vehicle was administered.

Determination of the intensity of the chronic stress effect Gustululations (Bhattacharya et al., 1987). On the 14th day (day 14), the rat was killed with a splashed neck, and its stomach was torn apart along a stunning curve and the number of ulcers noted. After histological confirmation, the severity of the ulcer changes: 0 = no ulcer; 1 = only the mucosal surface without clogging; 2 = half the thickness of the mucosa shows a change in necrosis; 4 = scored as complete destruction of mucosa with bleeding. The pooled ulcer score was then calculated according to Bhattacharya et al. (1987).

Adrenocortical hyperactivity activity Adrenal ascorbic acid (Zenker and Bernstein, 1958) and corticosterone concentrations (Selee, 1936), and plasma corticosterone levels (Selee, 1936) were determined and selected The effectiveness and strength of the stress technique was demonstrated.

Results and Discussion Chronic stress (CS) significantly increased the incidence, number and severity of gastric ulcers. All four test compounds had dose-related anti-ulcerogenic effects, even to varying degrees. The degree of the anti-ulcer generation effect was in the order of DCP>DBP> DHA≈EPA as shown in Table 7 below.

  Chronic stress (CS) is responsible for a significant decrease in corticosterone levels with adrenal ascorbic acid and accompanying substances and is increased in plasma corticosterone levels. These findings also suggest that the stress protocol used in this study induced significant stress. As expected, all four test compounds (EPA, DHA, DBP, DCP) reversed, to a different extent, these stress-induced adverse effects in dose-related approaches; There was no per se effect on the index of stress investigated in Table 8 as follows.

  The effects of DBP and DCP on suppression of humoral immunity in rat stress (Table 9) and rat cerebral frontal cortex SOD, CAT, GPx and LPO activity (Table 10) induced by chronic stress are the main Established a physiological contribution.

  The test drug was administered for 14 days in conjunction with stress measures.

  (Chi-square test). The animals bleed on day 14 (day 14) after sensitization with SRBC on day 1 (day 1).

Example 10
Effect of DCP on Arachidonic Acid Metabolism The anti-inflammatory effect of Shilajit and its main bioactive component, DCP, was evaluated using arachidonic acid (AA) metabolism. The effect of Shilajit on AA metabolism was tested on isolated human neutrophils. Both Shilajit and DCP are products of the AA-lipoxygenase pathway, so-called leukotriene-B ₄ (LTB ₄ ), 5-hydroxyeicosatetraenoic acid (5-HETE), 12-hydroxyeicosatetraenoic acid (12 -HETE) biosynthesis, and in a dose-dependent manner, the biosynthesis of cycloxygenase product, ie 12-hydroxyheptadecatrienoic acid (12-HHT). Whereas DCP was only 10 μg / ml, the greatest inhibitory effect was observed with Shilajit at a concentration of 50 μg / ml. The 1: 4 combination of 3,8-dihydroxydibenzo-alpha-pyrone (DBP) and fusom showed comparable activity at a concentration of 20 μg / ml. Shilajit fusom has been used as an effective and systemic drug delivery agent (Ghosal, 2003). These findings suggest that inhibition of leukotriene (and equivalent) synthesis by Shilajit and its main bioactive substance (DCP) is effective in treating therapeutics such as bronchial asthma.

  As shown in Tables 7 and 8, the above results show that while DCP is most active among Shilajit bioactive agents, DBP (3-hydroxy- and 3,8-dihydroxybenzo-alpha- A 1: 1 mixture of pyrone) suggests that it is more biologically active than one of its precursors, ie EPA (eicosapentaenoic acid) or DHA (docosahexaenoic acid). A similar graded effect of DBP and DCP was observed in chronic stress (CS) -induced perturbations in rat brain antioxidant enzymes and LPO activity (Table 9) and CS-induced suppression of humoral immunity in rats (Table 9). 10). The significance of DCP in biological systems and the fate of DCP after oral administration to experimental animals has also been shown.

Example 11
Antioxidant effect of DCP Inhibition of lipid peroxide (LPO) in rat brain by DBP and DCP induced by Fe-ADP-ascorbate Albino rats (Sprague Dawley strain) were sacrificed by cervical dislocation and decapitation. became. The brain was fully dissected and 10% (w / v) homogenate was prepared in 0.15 M KCl. The brain homogenate was centrifuged at 1500 rpm for 10 minutes and the supernatant was used for this study. Incubation mixtures contained test compounds dissolved in solvent at a final volume of 1 ml in brain homogenate (500 μl), distilled water (100 μl), or different concentrations (final volume 10-100 μg / ml). Peroxidation was initiated by adding FeCl ₃ (100 μM), ADP (1 mM) and ascorbate (100 μM), giving the final concentration as described above. After 30 minutes incubation at 37 ° C., the reaction was terminated by adding acetate buffer (1.5 ml, pH 3.5) and thiobarbituric acid solution (1.5 ml, pH 7.4) into 1 ml of LPO mixture. . The reaction mixture was heated at 85 ° C. for 30 minutes, cooled, centrifuged (2000 rpm for 10 minutes), and the absorbance of the supernatant was measured at 532 nm. IC ₅₀ values were calculated in the usual way by plotting the concentration of the test compound against the percentage inhibition of LPO.

Example 12
Metal Ion Chelating and Scavenging Effects on DCP This is the stability of metal ion complexes / conjugates on DCP and scavenging / chelating metal ions Determined by their capacity against.

The Schnitzer and Skinner ion exchange equilibration method (1966) was used for the determinations described above. In short, DCP amounts in the range of 10-50 mg were weighed in a 50 ml flask and dissolved in approximately 40 ml distilled water. To each flask, 5 ml of 1N-KCl solution was added. A 1 g quantity of K-saturated Dowex-50 resin (20-50 mesh, Bio-RAD Laboratories) was weighed in a 125 ml Erlenmeyer flask with a ground glass stopper. A solution containing the natural DCP-metal ion blend (Fe, Cu, Zn) was mixed with KCl, transferred to these flasks and stirred at 24 ± 1 ° C. for 1 hour. In a separate experiment, a known amount (about 500 μg) of an aqueous solution of MnCl ₂ , MoCl ₃ and WCl ₄ was added separately to the aqueous solution of DCP-KCl. The mixture was stirred as before and the stability constant was determined as follows. Thereafter, the exchange resin was removed by filtration. The filtrate and washing solution contain metal ions, ie Fe ²⁺ , Cu ²⁺ , Zn ²⁺ , which have the added metal ions, were analyzed by atomic absorption spectroscopy (Techtron AA-3 Atomic Absorption Spectrophotometer). .

At pH 3.5, the logarithmic stability constant for the different DCP-metal ion complexes was DCP-Cu, 3.44; DCP-Fe, 2.83; DCP-Zn, 1.47. The order of the stability of the different metal ions (expressed in decreasing order): Cu ²⁺ > Fe ²⁺ > Mn ³⁺ > Zn ²⁺ > Mo ³⁺ > W ⁴⁺ .

  As shown in Tables 7 and 8, the results show that while DCP is most active among Shilajit bioactive agents, DBP (3-hydroxy- and 3,8-dihydroxydibenzo-alpha-pyrones 1: 1 mixture) suggests that it is more biologically active than one of its precursors, EPA (eicosapentaenoic acid) or DHA (docosahexaenoic acid). Similar graded effects of DBP and DCP are associated with chronic stress (CS) -induced perturbations in rat brain antioxidant enzymes and LPO activity (Table 9), and CS-induced suppression of humoral immunity in rats. Observed in (Table 10). The antioxidant effect of DCP is evident as shown in Tables 11 and 12, which show the significance of DCP in biological systems and the fate of DCP after oral administration to experimental animals.

Example 13
Personal Care / Cosmetic Formulation A. Skin rejuvenation (O / W) lotion

Mix Procedure A, stir and heat to 65 ° C. Mix B, stir and heat to 65 ° C. Add A to B while stirring. Homogenize at moderate speed to avoid bubbling while cooling the mixture temperature to 40 ° C. Add C and homogenize. Gently stir until the mixture is homogeneous.
B. Sunscreen O / W Lotion (SPF 15)

Mix Method A, stir and heat to 80 ° C. Mix B, stir and heat to 80 ° C. Add A to B while stirring with a propeller mixer. Keep stirring A / B for 20 minutes while maintaining a temperature of 70-75 ° C. Mix C, stir until dissolved, and heat to 45 ° C. Add C to A / B with stirring. Qs water. Gently homogenize A / B / C and allow the mixture to cool to room temperature. Prepare using a high shear sprayer.
C. A liquid foundation having the following formulation was prepared by the following method.

Method A. The components (1) to (4) were mixed and dissolved together.
B. The components (9) to (12) were mixed with the aforementioned mixture A and mixed uniformly.
C. The components (5) to (8) were all dissolved uniformly, and the resulting mixture was maintained at 70 ° C.
D. The aforementioned mixture C was added to the aforementioned mixture B and mixed uniformly to give an emulsion.
E. After cooling the aforementioned mixture D, the components (13) to (15) were mixed therein to give a liquid foundation.
The liquid foundation prepared in Example 3 was found to have excellent stability over time. Application of this foundation to the skin can prevent the appearance of any wrinkles induced by the sun.
D. Moisture recovery body lotion

Method 1. Combine the ingredients in Phase A and heat to 80 ° C. for 45 minutes with mixing.

  2. Combine the ingredients in Phase B. Heat to 75-80 ° C and mix.

  3. Add Phase B to Phase A under homogenization. Homogenize until uniform.

  4). Add phase C to phases A and B. Remove from homomix and start cooling. Convert the propeller mix to 40 ° C.

  5. Add Phase D ingredients one at a time at 40 ° C. and mix well between each addition.

6). Add Phase E at 35 ° C. QS for water loss.
E. Hair gloss oil

Method 1. Add DCP to Lauryl lactate in a small mixing vessel. Heat the mixture at 60-70 ° C. Mix well with slow agitation until uniform. Cool to 30-35 ° C with stirring.

  2. Add alcohol to another larger container.

  3. When phase A is at 30-35 ° C, add phase A to the alcohol. Mix well until uniform. Add the remaining ingredients in turn throughout the mix, until uniform.

4). Add preservatives. Mix well until uniform.
F. Skin gloss / whitening lotion for face

Combine techniques A and heat to 70-75 ° C. Combine B and heat to 70-75 ° C. Add B to A while stirring. Add Phase C at 30 ° C. Use phase D to adjust the pH to 5.0-6.0. Add phase E. Mix until uniform.
Example 14
Pharmaceutical / Nutrition Formulation A Tablets and Capsules of the Invention

B. Antistress support tablet / capsule of the present invention

C. The cardiovascular support tablet of the present invention

D. Multi-vitamin and mineral supplement tablets of the present invention

Other raw materials and plant antioxidants: N-acetylcysteine, succinic acid (free form), choline (acid tartrate), inositol (hexanicotinate and inositol), N-acetylglucosamine, DMAE (acid tartrate) , N-acetyl L-tyrosine, coenzyme Q10, alpha lipoic acid, quercetin, Milk Thisle seed extract, grape seed extract, ginkgo leaf, bilberry extract.
E. Antidiabetic support tablets / capsules of the present invention

F. Weight loss support tablet of the present invention

G. Chewable tablet of the present invention

Blend all ingredients except Method 6 in a blender for 20 minutes. Select in 6 and blend for another 5 minutes. Press against a tablet using 7/16 with a standard concave tool.
H. The syrup of the present invention

I. Oral liquid of the present invention

J. et al. Snack bar using the present invention

K. Serial using the present invention

L. Beverage using the present invention

Example 15
Veterinary prescription Chewable tablet of the present invention

B. Vitamin tablets of the present invention (peanut butter flavor)

C. Granules of the present invention

D. Hematopoietic powder of the present invention

E. Liquid capsule of the present invention

F: Oral liquid of the present invention

G. Suspension of the present invention

H. Injection material of the present invention

The general structure of DCP and the complex set of DCP are shown. The general structure of DCP and the complex set of DCP are shown. Figure 5 shows DCP levels varying with time in erythrocytes of albino rats that assimilate DCP. The HPLC chromatogram of Shilajit DCP derived from the ammonium sulfate precipitate is shown. Figure 3 shows the relationship between 3,8-dihydroxydibenzo-alpha-pyrone and protein fraction.

Claims (38)

a. Dibenzo-alpha-pyrone or derivatives thereof;
b. Phosphocreatine;
c. A dye peptide having a molecular weight of about 2 KD or less; and d. Lipids having fatty acyl esters of glycerol,
A composition of dibenzo-alpha-pyrone chromoprotein (DCP) comprising: The composition of claim 1 comprising the dibenzo-alpha-pyrone of formula (I):

Wherein R ¹ is selected from the group consisting of H, OH, O-acyl and O-amino-acyl;
R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are each independently selected from the group consisting of H, OH, O-acyl, O-amino-acyl and fatty acid acyl groups.   The composition of claim 1, wherein the phosphocreatine is attached to the 3 or 8 position of the dibenzo-alpha-pyrone via an ester bond. The dye peptide is
One or more amino acids;
With carotenoids;
The composition according to claim 1, further comprising indigoid.   2. The composition of claim 1, wherein the chromoprotein has a molecular weight of about 2 to about 20 KD.   6. The composition of claim 5, wherein the chromoprotein comprises one or more amino acids selected from the group consisting of methionine, arginine, glycine, alanine, threonine, serine, proline and hydroxyproline.   The composition of claim 1, wherein the pigment peptide comprises a carotenoid moiety, wherein the carotenoid moiety is astaxanthin and equivalents. Lipid is a saturated or unsaturated fatty acids having a carbon chain length of from about C ₁₄ -C _24, The composition of claim 1.   The composition according to claim 8, wherein the polyunsaturated fatty acid substituent has an unsaturation degree of 1-6.   The composition according to claim 9, wherein the polyunsaturated fatty acid is eicosapentaenoic acid and / or docosahexaenoic acid.   The composition of claim 1, wherein the DCP further comprises iron, calcium, copper, zinc, magnesium, vanadium, and / or metal ions in a range of about 1 to about 500 ppm level.   The composition of claim 1, wherein the DCP further comprises a low molecular weight ligand.   A skin care, hair care, pharmaceutical, or nutritional or veterinary formulation comprising the composition of claim 1 present in an amount of about 0.05 to about 50% by weight.   14. A skin care or protective formulation according to claim 13, in the form of a lotion, cream, gel or spray, wherein the composition is present in an amount of about 0.05 to about 5% by weight.   14. A pharmaceutical formulation according to claim 13 in the form of a tablet, syrup, elixir or capsule.   14. The nutritional formulation of claim 13, comprising from about 0.5 to about 30% of the composition.   Cosmetically acceptable carriers and the group consisting of sunscreen, antioxidant, preservative, self-tanning agent, perfume, oil, wax, high pressure gas, waterproofing agent, emulsifier, thickener, moisturizer and emollient 15. A skin care or protective formulation according to claim 14 further comprising one or more cosmetic adjuvants selected from.   The pharmaceutical formulation according to claim 15, further comprising a pharmaceutically acceptable carrier.   The nutritional formulation of claim 16 further comprising a nutritionally acceptable carrier. A method of isolating a DCP composition according to claim 1 from a Shilajit composition comprising at least 0.5% to 10% w / w dibenzo-alpha-pyrone chromoprotein, comprising:
1) pulverizing the natural Shilajit lock substance, extracting sequentially with hot ethyl acetate and methanol and removing the soluble low and medium molecular weight organic compounds by filtration;
2) triturating the ethyl acetate and methanol insoluble material with hot water followed by pH 5.0 citrate buffer;
3) filtering the combined extraction mixture to remove insoluble materials including polymerized humic materials, minerals and metal ion salts;
4) Gradually saturate the combined aqueous filtrates with increasing concentrations of ammonium sulfate to obtain a purple brown precipitate of the mixture of DCPs, or concentrate the combined aqueous solution and add acetone to make the DCP brownish Depositing as a red or off-white precipitate, filtering the DCP, and evaporating the filtrate to obtain a more complex mixture of less complex DCP;
5) fractionating the purple-brown solid residue resulting from ammonium sulfate saturation by gel filtration of Sephadex and electrophoresis and isolating the DCP composition from Shilajit;
Including a method.   21. The method of claim 20, wherein the Shilajit composition is about 12% to about 40% w / w dibenzo-alpha-pyrone chromoprotein. A method for isolating the DCP composition of claim 1 from an ammonite fossil comprising:
1) pulverizing the ammonite fossil material, extracting sequentially with hot ethyl acetate and methanol and removing soluble low and medium molecular weight organic compounds by filtration;
2) triturating the ethyl acetate and methanol insoluble material with 0.1 N HCl;
3) filtering the aqueous acid extract to remove insoluble material, including polymerized humic material, and dissolving in a minimum amount of water;
4) Gradually saturate the aqueous solution with increasing concentrations of ammonium sulfate to obtain a purple brown precipitate of the mixture of DCP, or concentrate the combined aqueous solution and add acetone to make the DCP brownish red or Depositing as an off-white precipitate, filtering the DCP, and evaporating the filtrate to obtain a more complex mixture of less complex DCP;
5) fractionating the purple-brown solid residue resulting from ammonium sulfate saturation by gel filtration of Sephadex and electrophoresis, and isolating the DCP composition from the ammonite fossils;
Including a method. A method for isolating a DCP composition according to claim 1 from coral fossils comprising:
1) pulverizing the coral fossil material, extracting sequentially with hot ethyl acetate and methanol, and removing the soluble low and medium molecular weight organic compounds by filtration;
2) triturating the ethyl acetate and methanol insoluble material with 0.1 N HCl;
3) filtering the aqueous acid extract to remove insoluble material, including polymerized humic material, and dissolving in a minimum amount of water;
4) Gradually saturate the aqueous solution with increasing concentrations of ammonium sulfate to obtain a purple brown precipitate of the mixture of DCP, or concentrate the combined aqueous solution and add acetone to make the DCP brownish red or Depositing as an off-white precipitate, filtering the DCP, and evaporating the filtrate to obtain a more complex mixture of less complex DCP;
5) fractionating the purple-brown solid residue resulting from ammonium sulfate saturation by gel filtration of Sephadex and electrophoresis, and isolating the DCP composition from coral fossils;
Including a method. A method for isolating a DCP composition according to claim 1 from an invertebrate, comprising:
1) extracting body fresh with hot ethyl acetate to remove low molecular weight free organic compounds and lipids as soluble fractions;
2) extracting the ethyl acetate with a Brid-Dyer solvent system;
3) evaporating and concentrating the Brid-Dyer solvent extract under reduced pressure and dissolving in a minimum amount of water;
4) Gradually saturate the water with increasing concentrations of ammonium sulfate to obtain a purple brown precipitate of the mixture of DCPs, or concentrate the combined aqueous solution and add acetone to make the DCP brownish red or Depositing as an off-white precipitate, filtering the DCP, and evaporating the filtrate to obtain a more complex mixture of less complex DCP;
5) fractionating the purple-brown solid residue resulting from ammonium sulfate saturation by gel filtration of Sephadex and electrophoresis, and isolating the DCP composition from the invertebrates;
Including a method.   A method of treating chronic stress comprising administering to a patient in need thereof a therapeutically effective amount of the composition of claim 1. A composition comprising dibenzo-alpha-pyrone chromoprotein (DCP) of formula (II):

Wherein R ¹ is selected from the group consisting of H, OH, O-acyl and O-amino-acyl;
R ² is selected from H and CH ₃ ;
R ³ is selected from H and fatty acids;
R ⁴ is selected from H and fatty acids; and R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are each independently H, OH, O-acyl, O-amino-acyl and Selected from the group consisting of fatty acid groups.   27. The composition of claim 26, wherein the DCP further comprises iron, calcium, copper, zinc, magnesium, and / or metal ions in the range of about 1-500 ppm level.   28. The composition of claim 27, wherein the DCP further comprises a low molecular weight ligand.   27. A skin care, hair care, pharmaceutical or nutritional formulation comprising the composition of claim 26 present in an amount of about 0.05 to 50% by weight.   27. A method of treating chronic stress disease comprising administering to a patient in need thereof a therapeutically effective amount of the composition of claim 26.   14. A method of increasing the cognitive effect of learning comprising administering to a patient in need thereof a therapeutically effective amount of the composition of claim 13.   14. A method of treating stress disorders comprising administering to a patient in need thereof a therapeutically effective amount of the composition of claim 13.   The disease is selected from stress induced by anxiety, depression-induced stress, temperature-induced stress, gastric ulcer-induced stress, seizure-induced stress, and adrenal cortex-induced stress 35. The method of claim 32.   14. An antioxidant defense selected from the group consisting of superoxide dismutase (SOD), catalase and glutathione peroxidase comprising administering to a patient in need thereof a therapeutically effective amount of the composition of claim 13. A method of regulating the immune system by increasing the number of enzymes.   30. A method of increasing the cognitive effect of learning comprising administering to a patient in need thereof a therapeutically effective amount of the composition of claim 29.   30. A method of treating stress disorders comprising administering to a patient in need thereof a therapeutically effective amount of the composition of claim 29.   The disease is selected from stress induced by anxiety, depression-induced stress, temperature-induced stress, gastric ulcer-induced stress, seizure-induced stress, and adrenal cortex-induced stress 38. The method of claim 36.   30. An antioxidant defense selected from the group consisting of superoxide dismutase (SOD), catalase and glutathione peroxidase, comprising administering to a patient in need thereof a therapeutically effective amount of the composition of claim 29. A method of regulating the immune system by increasing the number of enzymes.

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