Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
  • A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
  • A61K8/498—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/02—Nutrients, e.g. vitamins, minerals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
  • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q5/00—Preparations for care of the hair

Definitions

• This invention relates to the composition of oxygenated dibenzo-alpha-pyrone chromoproteins (DCP) and their isolation from shilajit, fossils of ammonites, corals and other invertebrates. More particularly, the invention relates to the description of DCP-composition comprising oxygenated dibenzo-alpha-pyrone or its conjugates, phosphocreatine, proteins, fatty acyl esters of glycerol and other small ligands, e.g.,carotenoids, sterols and aromatic acids, as core structural fragments, and their biological functions. Pharmaceutical, nutritional, veterinary, skin care and personal care formulations are also described. These findings establish DCPs as the major bioactives of shilajit.
• the present invention describes one such class of pigmented proteins, named dibenzo-alpha-pyronechromoproteins (abbreviated as DCPs), isolated in large abundance, from shilajit, fossils of ammonites, corals and other marine invertebrates. Summary of the Invention The present invention relates to compositions of DCPs, isolation, and their use in treating various adaptogenic conditions, such as chronic stress.
• DCPs dibenzo-alpha-pyronechromoproteins
• the invention provides a composition of dibenzo-alpha- pyrone-chromoproteins (DCPs) which include dibenzo-alpha-pyrone or their derivatives; Phosphocreatine; Chromo-peptides of molecular weights of ⁇ 2 KD; and Lipids having fatty acyl esters of glycerol.
• DCPs dibenzo-alpha-pyrone-chromoproteins
• Another embodiment of the invention includes dibenzo-alpha-pyrones of formula (I)
• 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 independently selected from the group consisting of H, OH, O-acyl, O-amino-acyl, and fatty acyl groups.
• Another embodiment of the invention includes a composition wherein phosphocreatine is attached to the 3- or 8-position of said dibenzo-alpha-pyrones via an ester linkage.
• the chromo-peptides include one or more amino acids; carotenoids; and indigoids.
• the chromo-proteins have a molecular weight of about 2 to about 20 KD.
• Another embodiment of the invention provides a skin care, hair care, pharmaceutical, veterinary or nutritional formulation comprising a DCP composition present in an amount of about 0.05% to about 50% by weight.
• the skin care or protection formulation can be 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 pharmaceutical formulation comprising a DCP composition wherein the pharmaceutical formulation is in the form of a tablet, syrup, elixir or capsule.
• a nutritional formulation comprising a DCP composition wherein the nutritional formulation contains about 0.5% to about 30% of the DCP composition by weight.
• a veterinary formulation comprising a DCP composition wherein the veterinary formulation contains about 0.5% to about 30% of the DCP composition by weight.
• Another embodiment of the invention provides a process for isolating DCP compositions from shilajit compositions comprising about 0.5% to about 10% w/w dibenzo-alpha-pyronechromoproteins, the process includes the steps of l)extracting shilajit successively with hot ethyl acetate and methanol to remove the soluble low and medium molecular weight organic compounds by filtration; 2) triturating the ethyl acetate and methanol insoluble material with hot water and then citrate buffer of pH 5.0; 3) filtering the combined extract-mixture to remove insoluble substances comprising polymeric humic materials, minerals and metal ion salts; 4) gradually saturating the combined aqueous filtrate with increasing concentrations of ammonium sulphate to obtain purple-brown precipitate of mixture of DCPs, or concentrating the combined aqueous solution and adding acetone to precipitate DCPs as brownish-red or off-white precipitate and filtering the DCPs and evaporating
• FIG. 1A and IB show the general structure of DCPs and the conjugate assembly of DCPs.
• Fig. 2 shows changes in different DCP levels with time in red blood cells of DCP-fed albino rats.
• Fig. 3 shows HPLC chromatograms of Shilajit DCPs from ammonium sulphate precipitations.
• DCPs comprising organo-mineral constituents exhibit orange, purple and yellow colors contributed by oxygenated carotenoids known as xanthophylls and indigoids derived from systemic oxidation of tryptophan moieties.
• the DCPs of shilajit exhibit absorption maxima in the UV and visible regions at ⁇ -225, -275, - 320, - 392, - 470, - 492, 500-535, 620-660 nm.
• the conjugated proteins containing both less polar and more polar fractions still retained some of the amino acids/ small peptides, xanthophylls and indigoids, as determined by HPLC of the degraded products, in the lipase degradation products and some amino acid/ small peptide in the conjugate DBPs even after classical acidic hydrolysis.
• DCPs produced free DBPs and small conjugated DBP metabolites, fatty acids and amino acids.
• the facile removal of the acylated compounds by saponification suggested that some aminoacyl and fatty acyl moieties are attached to the phenolic hydroxyl group(s) of DBPs.
• the occxurence of small O-acyl conjugates of amino acids in 3-OH-DBP from 3-O-acyl glycinoyl and 3-O-acyl arginoyl DBPs, and also creatine in DCPs support the DBP-prosthetic group structure of the DCPs shown in Formula 1.
• R , ⁇ H, OH, O-acyl, O-amino acyl, or di- or tri-peptides of these aminoacids;
• R 2 H or CH 3 ;
• R 3 H or C ⁇ 4 - C 24 saturated or unsaturated fatty acid; degree of unsaturation ranging from one to six;
• R 4 H or C 14 - C 2 saturated or unsaturated fatty acid; degree of unsaturation ranging from one to six;
• 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 chromo-proteins have a weight of 2-20 kilodaltons (KD), and include but are not limited to amino acids, di- and tri-peptides of these aminoacids, carotenoids and indigoids.
• KD kilodaltons
• Acyclic and cyclic carotenoids or xanthophylls and indigoids, such as lutein, astaxanthin, and beta-carotene are pigments.
• Fatty acids may be branched or unbranched and contain carbon atoms between 14 and 24, and may be either saturated or unsaturated. The degree of unsaturation is between one and six. Degree of unsaturation is the number of double bonds present.
• Acyl is -COR where R may be branched or unbranched and contain carbon atoms between 16 and 18, and may 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 results when an amide bond is formed between the -NH 2 of one amino acid and the -COOH of a second amino acid; a tripeptide results from linkage of three amino acids via two amide bonds, and so on. Any number of amino acids can link together to form large chains.
• the numbering pattern of the dibenzo-alpha-pyrone is as follows:
• DCPs contain proteins of molecular weight with a range between 2 to about 20
• DCPs in which the apoprotein is colorless, and the colored compounds containing long prosthetic groups (e.g., DBPs and lipids), can be dissociated by simple treatment of aqueous solution of DCPs, either with acetone or ethyl alcohol.
• the colorless apoproteins exhibit simple HPLC patterns and on acid hydrolysis produced, apart from DBPs and conjugates, the amino acids described above.
• DCPs isolated from fossils of Ammonites, are readily split into the colorless apoproteins and coloring matter, which are soluble in the extracted organic solvents. 2.
• the other class constitutes DCPs in which the coloring matter comprising carotenoids and indigoids are ordinarily undissociable from the apoprotein.
• This class of DCPs was isolated from shilajit and from some rare species of fossils of Ammonites (e.g., Perisphinctes with red protoconch) Proteins of some invertebrates spread at the air/water interface with extreme reluctance.
• the apoproteins when dissociated from the prosthetic groups (e.g., containing the coloring matter such as carotenoids), spread smoothly during electrophoresis.
• the carotenoids in such chromo-proteins seem to act as a 'lock' on the tertiary or quaternary structure of the proteins against denaturation.
• the colorless apoproteins formed from dissociation of chromoproteins, by contrast undergo immediate coagulation and partial denaturation.
• shilajit-DCPs the association of the chromo-molecules and the apoproteins are not, ordinarily, dissociable. A specific, tenacious, combination of the two moieties is conceivable. Consistent with this postulate, the chromo-compounds in shilajit-DCPs were found to be associated with both the lipid and apoprotein fractions.
• lipid moieties present in the Formula 1 are covalently linked with the prosthetic group(s). This was suggested by the following study. Exhaustive extractions of DCPs by Bligh and Dyer solvent system, suitable for extraction of lipids, did not yield any free fatty acid but gave a small amount of acylated DCPs. The major insoluble residue on reaction with lipase produced Cj4 to C24 fatty acids in which C j g. ⁇ , C j g-Q and Cig- j were the main components as depicted in Table 1. Thus, lipoproteins seem to constitute an integral part of the DCPs. Table- 1 : Fatty acids composition of four ammonium sulphate precipitated DCPs after Lipase cut.
• a+b 100% of total fatty acids.
• c Expressed as % of total fatty acids present in each sample.
• Many of the shilaj it-bearing mountains have been found to be rich storehouses of marine invertebrate fossils, such as of the phyla of Arthropoda, Brachiopoda and Mollusca, of the Phanerozoic era. This co-occunence of shilajit and the invertebrate fossils, as depicted in Table 2, is a consistent phenomenon.
• acyl moiety was constituted of C ⁇ 6-C20 fatty acids Q quenching mode D deuterium lamp, wave length 260 nm M mercury lamp, wave length 360 nm; F, fluorescence mode T tungsten lamp, wave length 520 nm; BMP, benzidine-metaperiodate staining reagent for polyols, sugars; Nin, ninhydrin reagent for detection of amino acids
• DBPs Oxygenated dibenzo-alpha-pyrones (DBPs), on being synthesized in the animal living systems from EPA, are transformed into several DBP-co ⁇ jugates (HPLC-t R : 2.31, 2.99, 3.46 and 3.86 min). These components were also detected in DCPs, isolated from shilajit. A dynamic turnover of these constituents was observed (FIGURE 2) on oral administration of DCPs (200 mg/Kg b.w.) to albino rats, followed by HPLC analysis of the constituents in the conesponding RBC.
• DBPs Oxygenated dibenzo-alpha-pyrones
• DCPs which are also the constituents of animal tissues, act in the form of enzymes and hormones in regulating and fulfilling several biological functions. DCPs may participate in a variety of functions in the producer organisms including protective-colorations which provide protection from radiation, electron transport, and enzyme activity and in their sustenance and development. DCPs, which have transport properties like those of the fulvic acids (FAs) of shilajit, can enter into recipient cells and elicit biological responses much more pronounced than free DBPs. Extensive pharmacological and immunological evaluations of DCPs have now demonstrated them to be 2-5 times more potent than any of the other constituents of shilajit as adaptogen and immunomodulator.
• FAs fulvic acids
• Arginine phosphate plays an important role in the storage of energy in invertebrates; the same role is played by creatine produced from a combination of argininephosphate and glycine phosphate in vertebrates.
• Creatine phosphate and arginine phosphate are reserves of phosphates of high energetic potential and, hence, the name 'phosphagens' given to these compounds as shown in Scheme 1.
• ATP Synthesis catalysed by shilajit
• ADP ADP
• Creatine/Arginine Phosphagens (in shilajit-DCPs) Scheme 1
• An energetic coupling represents the energy storage reaction when ATP is present in excess and, inversely, the formation of ATP by the reverse reaction when the cells need the ATP.
• DBP- phosphagen complexes in living organisms as the indices of their energy status, then in the event of dearth of these phosphagens, administration (p. o.) of shilajit would replenish them.
• the chromoproteins (DCPs), participate in a wide variety of functions in animal biological systems.
• DCPs have been encountered in the lowest form of animal organisms (foraminifera, in other marine invertebrates, and in haemo lymph of termites), in higher animals (rodents, beaver, chimpanzee, sheep), and in man. DCPs participate in electron transport [systemic ATP synthesis by DCPs is conceivable because oral administration of DBP produced creatine and conjugated product(s)] and oxido-reductase reactions; catalyze other enzyme activities (e.g., ATPase function as described in Cheesman, 1967); the larger abundance of DCPs in female invertebrate fossils of the Jurassic (e.
• DCPs chromo-molecules in DCPs were found associated with both the lipid as well as the protein fractions of the complex molecules. 4. Development of embryos in invertebrates require carotenoproteins. 5. As simulator/sunogates of bio-energetics, e.g., ATP; creatine synthesis. 6. Immuno-modulator. 7. Captivators of oxidative free radicals, Reactive Oxygen Species (ROS), Reactive Nitrogen Species (RNS). 8. Scavengers/chelators of loose metal ions (Fe, Cu, Mn, W). 9. DCPs play a crucial vitalizer role in all organisms since the evolution of life on Earth.
• ROS Reactive Oxygen Species
• RNS Reactive Nitrogen Species
• the features of the isolation and use of DCPs provides 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 protection formulation can be 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.
• the features of the invention provide a pharmaceutical formulation comprising a DCP composition wherein the pharmaceutical formulation is in the form of a tablet, syrup, elixir or capsule.
• the features of the invention provides a nutritional formulation comprising a DCP composition wherein the nutritional formulation contains about 0.5% to about 30%) of the DCP composition by weight.
• the features of the invention provides a veterinary formulation comprising a DCP composition wherein the veterinary formulation contains about 0.5% to about 30% of the DCP composition by weight.
• the features of the invention provides a process for isolating DCP compositions from shilajit compositions comprising about 0.5% to about 10% w/w dibenzo-alpha- pyronechromoproteins, the process includes the steps of l)extracting shilajit successively with hot ethyl acetate and methanol to remove the soluble low and medium molecular weight organic compounds by filtration; 2) triturating the ethyl acetate and methanol insoluble material with hot water and then citrate buffer of pH 5.0; 3) filtering the combined extract-mixture to remove insoluble substances comprising polymeric humic materials, minerals and metal ion salts; 4) gradually saturating the combined aqueous filtrate with increasing concentrations of ammonium sulphate to obtain purple-brown precipitate of mixture of DCPs, or
• the features of the invention provides similar processes for extracting and isolating DCPs from fossils of ammonites, fossils of corals, and from invertebrates.
• the features provide a method for treating chronic stress disorders, including administering to a patient in need thereof a therapeutically effective amount of a DCP composition and a method for increasing cognition learning which includes administering a DCP composition.
• the following examples will serve to further typify the nature of the invention.
• the marc was analysed for inorganic minerals and humic substances.
• the aqueous solution was differently saturated with ammonium sulfate (25%, 50%, 75% and 100%)) when DCPs of different complexities were precipitated as purple-brown solid.
• the solid residues were subjected to Sephadex gel filtration and electrophoresis for further purification of DCPs.
• the same general procedure was followed for the isolation of DCPs from the marine samples.
• one variation constituted addition of acetone, instead of ammonium sulfate and to isolate DCPs from acetone-insoluble and soluble fractions in the usual way.
• EXAMPLE 2 Extraction and isolation of DCPs of marine invertebrate fossils (general procedure) In a typical experiment, fossils of Nummulites (foraminifera, GSI type
• the other part was subjected to lipase reaction (see EXAMPLE 5).
• the DCP-I compound was obtained as a pink colored powder; pH (1% aqueous solution) 8.02; N, 17.8%; metal ions (in ppm) Fe, 186.3; Cu, 8.8; Zn, 23.4.
• the DCP-II compound was 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 canied out by Sephadex ion exchange on DEAE-Sephadex G-50, using phosphate buffer (pH 7.2).
• t R 1.5 min signal (Fig. 3) suggested that higher Mw proteins, like B-48, might also occur in DCPs.
• adherent ligands particularly DBPs.
• Another observation was the association of DBPs as ligands in DCPs (Fig. 4).
• PR-25, -50, -75 and -100 denote respective ammonium sulfate precipitated protein fractions. Note that in the PR-50 and -75, the abundances of 3,8- dihydroxydibenzo-alpha-pyrone are very high suggesting that the DBPs are preferentially associated with low/medium MW hpoproteins.
• EXAMPLE 6 Determination of amino acids The mixture of amino acids produced in the acidic hydrolysates of DCPs was converted into trimethylsilyl derivatives (O-/N-TMS) and then subjected to GC-MS analysis by using conesponding markers, similarly prepared with the standard amino acids.
• EXAMPLE 7 Determination of creatine This method, based on the color reaction developed by creatine in the presence of diacetyl and ⁇ -napthol, was described by Banett (1936).
• Test compounds and Vehicles EPA (Aldrich, Milw.), DHA (Sigma), DBPs and DCPs were separately suspended/ dissolved in 0.3% carboxymethylcellulose(CMC) in distilled water and administered orally (p.o.) , for 14 days , starting on day 1, 60 min prior to electroshock. Control animals received only the vehicle in either unstressed or the stressed rats for the same period in a volume of 2.5ml/kg, p.o. Estimations were conducted on day 14, one hour after the last stress procedure and two hours after the last test compound or vehicle was administered. Determination of intensity of chronic stress effects Gastric ulcerations (Bhattacharya et al., 1987).
• Adrenocorticoid activity Adrenal gland ascorbic acid (Zenker and Bernstein, 1958) and corticosterone concentrations (Selye, 1936), and plasma corticosterone levels (Selye, 1936) were determined to substantiate the validity and intensity of the stress procedure adopted.
• Results and discussion Chronic stress (CS) significantly increased the incidence, number and severity of gastric ulcers. All the four test compounds had, albeit in different degrees, dose- related anti- ulcerogenic effect. The extent of the anti-ulcerogenic effect was in the order: DCPs>DBPs>DHA-EPA as follows in Table-7. Table-7: Effects of shilajit constituents on chronic stress (CS) induced gastric ulceration in albino rats.
• b p ⁇ 0.05 vs vehicle control group.
• c p ⁇ 0.05 vs chronic stress group (CS).
• SOD superoxide dismutase
• CAT catalase
• GPx glutathione peroxidase
• LPO lipid peroxidation Test drugs were administered 14 days concomitant with stress procedure.
• Table-10 Effects of DBPS and DCPs on chronic stress-induced suppression of humoral immunity in rats 8 .
• Shilajit and DCPs both inhibited the biosynthesis of AA-lipoxygenase pathway products, namely, leukotriene-B 4 (LTB 4 ), 5-hydroxyeicosatetraenoic acid (5-HETE), 12- hydroxyeicosatefraenoic acid (12-HETE) and also inhibited the biosynthesis of the cycloxygenase product, 12-hydroxyheptadecatrienoic acid (12-HHT), in a dose dependant manner.
• Maximum inhibitory effects were observed at a concentration of 50 ⁇ g/ml of shilajit, while in case of DCPs, it was only 10 ⁇ g/ml.
• DCPs major bioactives
• DBPs (1:1 mixture of 3- hydroxy - and 3,8 -dihydroxydibenzo-alpha-pyrone
• DBPs are biologically more active than either of its precursors, namely, EPA (eicosapentaenoic acid) or DHA (docosahexaenoic acid) while DCPs are the most active among the bioactive agents of shilajit.
• EPA eicosapentaenoic acid
• DHA docosahexaenoic acid
• Similar graded effects of DBPs and DCPs were observed on chronic stress (CS)-induced perturbations in rat brain antioxidant enzymes and LPO activities (Table-9) and CS-induced suppression of humoral immunity in rats (Table- 10).
• the incubation mixture contained in a final volume of 1 ml., brain homogenate (500 ⁇ l), distilled water (100 ⁇ l) or test compounds dissolved in solvents at different concentrations (10 to 100 ⁇ g/ml of the final volume). Peroxidation was initiated by adding FeC ⁇ (100 ⁇ M), ADP (1 mM) and ascorbate (100 ⁇ M) to give the final concentration stated. After incubating at
• DCPs and their capacity to scavenging/chelating loose metal ions were used for the abovementioned determination. Briefly, amounts of DCPs ranging from 10 to 50 mg were weighed into 50 ml volumetric flasks and dissolved in approximately 40 ml of distilled water. To each flask, 5 ml of 1N-KC1 solution was added. One-gram quantities of K-saturated Dowex-50 resin (20-50 mesh, Bio-RAD Laboratories) were weighed into 125 ml of ground glass-stoppered Erlenmeyer flasks.
• Atomic Absorption Spectroscopy Techtron AA-3 Atomic Absorption Spectrophotometer.
• log stability constants for the different DCP-metal ion complexes were: DCP-Cu, 3.44; DCP-Fe, 2.83; DCP-Zn, 1.47.
• Procedure Combine A, stir and heat to 65° C. Combine B, stir and heat to 65 ° C. Add A to B while stirring. Homogenize at moderate speeds to avoid foaming, while allowing mixture temperature to cool to 40° C. Add C, homogenize. Stir gently until mixture is homogenous.
• Procedure Combine A, stir and heat to 80° C. Heat B to 80° C. Add A to B while stirring with a propeller mixer. Continue stirring A/B for 20 minutes while maintaining the temperature between 70-75° C. Combine C, heat and stir to 45° C until dissolved. Add C to A/B with agitation. Qs water. Gently homogenize A/B/C allowing mixture to cool to room temperature. Adjust pH to -6.5, if necessary, with TEA. Use high shear spray device to dispense.
• a liquid foundation having the following formulation was prepared according to the following method.
• Palmitic Acid and) Stearic Acid (and) Lecithin
• the target weight of tablet/capsule is 400 mg; Avicel pH 101 and Starch may be adjusted suitably to reach the target weight.
• the blended material can be filled into appropriate capsules.
• Vitamin A (beta carotene) 25,000 IU 3. Vitamin A (palmitate) 10,000 IU 4. Vitamin B- 1 (Thiamin Nitrate) 10 mg 5. Vitamin B-2 (Riboflavin) 10 mg 6. Inositol Hexanicotinate, Niacinamide & Niacin 20 mg 7. Vitamin B-5 (Calcium D-Pantothenate) 10 mg 8. Vitamin B-6 ((Phyridoxine HCL) 10 mg 9. Vitamin B-l 2 (Cyanocobalamin) 200 meg 10. Biotin 500 meg 11. Folic Acid 800 meg 12. Vitamin C 180 mg (Magnesium, Manganese & Zinc Ascorbates) 13.
• Vitamin C 20 mg (from 476 mg of Ascorbyl Palmitate) 14.
• Vitamin D-3 Cholecalciferol 400 IU 15.
• Vitamin E D-alpha Tocopheryl (Natural) 600 IU 16.
• Boron Amino Acid Chelate) 2 mg 17.
• Calcium Succinate, Carbonate, Malate) 20 mg 18.
• Copper (Sebacate) 1 mg 19.
• Iodine from Kelp
• Manganese (Ascorbate) 30 mg 21.
• DCPs 0.10-50.00% by weight 2.
• Vitamin B-6 (as Pyridoxine HCI) 10 mg 3.
• DCPs 0.10-50.00% by volume 2.
• Purified Water q. s. 3.
• Excipients Preservatives, stabilizers, q. s. sweetners, flavors, colors, etc.
• Vitamin B Tricalcium Phosphate and q.s Calcium Carbonate
• Magnesium Magnesium Oxide
• Vitamin A Vitamin C
• Vitamin D-3 Vitamin B-l (Thiamin)
• Vitamin B-2 Vitamin B-2 (Riboflavin)
• Vitamin B-6 Pyridoxine
• Vitamin B-l 2 Cyanocobalamin
• Natural Vitamin Acetate
• Niacin Biotin, Pantothenic Acid, Zinc, Folic Acid, Vitamin K, Selenium.
• Protein Blend Soy protein isolate, Hydrolyzed collagen, Whey protein isolate, Calcium/Sodium Caseinate ), Glycerine, Polydexfrose (fiber), Water, Cocoa Butter, Natural Coconut Oil (non-hydronated), coconut, Cellulose, Cocoa Powder, Olive Oil, Lecithin, Natural and Artificial Flavor, Maltodextrin, Guar Gum, Citric Acid (Flavor Enhancer), Sucralose K.
• Ingredient Ingredient Ingredient Quantity per 1 Kg No. 1 DCPs 0.50-30.00%) by weight 2 Excipients: Whole Grain Oats, Oat Bran, q.s Sugar, Modified Corn Starch, Brown Sugar Syrup, Salt, Calcium Carbonate, Trisodium Phosphate, Wheat Flour, Vitamin E (Mixed tocopherols), Zinc & Iron (Mineral nutrients), Niacinamide (A B Vitamins), Vitamin B6 (Pyridoxine Hcl), Vitamin B2 (Riboflavin), Vitamin Bl (Thiamin Mononitrate), Vitamin A (Palmitate), Vitamin A B (Folic acid), Vitamin B12, Vitamin D
• Ingredient Ingredient Quantity per No. 500 mL 1 DCPs 0.50-30.00% by volume Excipients: Filtered Water, Food Starch- q.s Modified, Citric Acid, Bitter Orange, Green Tea Extract, Maltodextrin, Whey Protein Isolate, High Fructose Com Syrup and/or Sucrose and/or Sugar, Sodium Benzoate, Caffeine, Niacin, Glycerol Ester of Wood resin, Flavors, Colors
• DCPs 0.10-50.00% by weight 2.
• Other Ingredients q. s. Brewer's Yeast Powder, Garlic, Whey, Beef Liver, Peanut Butter, Silica Gel, Niacin, Riboflavin, Thiamine Mononitrate, Ascorbic acid
• DCPs 0.10-50.00% by weight 2.
• Other Ingredients q.s. Potassium Gluconate, Wheat, Sucrose, Hydrolyzed Vegetable Protein, Silicone Dioxide, TBHQ (preservative)
• DCPs 0.10-50.00% by weight 2.
• Other Ingredients q.s. Heme iron polypeptide, Niacin (Vitamin B3), Vitamin E acetate, Riboflavin (Vitamin B2), Thiamine (Vitamin Bl), Pyridoxine (Vitamin B6), Vitamin B12, Copper Sulfate, Cobalt sulfate, Soybean oil, Whey, Natural sweet apple and molasses flavors
• the capsules may be punctured and the liquid contents squeezed onto food, if desired.
• DCPs 0.10-50.00% by weight 2.
• Other Ingredients q. s. Safflower Oil, Gelatin, Fish Oil, Glycerin, Borage Seed Oil, Vitamin E, Water
• DCPs 0.10-50.00% by volume 2.
• Excipients Preservatives, stabilizers, q.s. sweeteners, flavors, colors, etc.

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• Coloring Foods And Improving Nutritive Qualities (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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• 2005
  • 2005-03-14 EP EP05779897A patent/EP1750739A4/de not_active Withdrawn
  • 2005-03-14 JP JP2007508354A patent/JP2008504231A/ja active Pending
  • 2005-03-14 CA CA002562829A patent/CA2562829A1/en not_active Abandoned
  • 2005-03-14 WO PCT/US2005/008577 patent/WO2005099739A1/en active Application Filing

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