Classifications

• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
  • C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
  • C07D309/08—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D309/10—Oxygen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
  • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
  • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan

Definitions

• Embodiments of the invention relate to a method for simply and efficiently isolating and stabilizing ultra low molecular weight aminoglycans from waste egg shells.
• Embodiments of the invention relate to processes for isolating, stabilizing and formulating low molecular weight aminoglycans from waste egg shells.
• the aminolycan extract is useful for the preparation of cosmetic creams with skin moisturizing and anti-wrinkle properties.
• Balazs et al (U.S. Pat. No. 4,141,973) has described a process to isolated pure hyaluronic acid from animal tissue bearing molecular weights in the range of 1 MD to 6 MD useful as a replacement for synovial fluids and vitreous humor.
• Stahl et al (U.S. Pat. No. 6,537,795) have described a process to produce and isolate aminoglycans from cultivated strains of streptococci fermentation. These aminoglycans are characterized by extreme high molecular weights above 6 MD and are useful for cartilage replacement therapy.
• Embodiments of the invention relate more particularly to step (b), wherein the aqueous polar salt solution can be the sodium, potassium, calcium or magnesium salts of citrate, glutamate, acetate, pyrrolidonecarbonate, tartrate, glycinate, sulfate, sulfite, nitrate, carbonate, oxalate to yield a solution containing aminoglycan compound of formula I wherein M may be one or more instances of Na, Ca, K, Mg and n is an integer between 20 and 40, which is suitable for selective gelation and isolation.
• the aqueous polar salt solution can be the sodium, potassium, calcium or magnesium salts of citrate, glutamate, acetate, pyrrolidonecarbonate, tartrate, glycinate, sulfate, sulfite, nitrate, carbonate, oxalate to yield a solution containing aminoglycan compound of formula I wherein M may be one or more instances of Na, Ca,
• the aminoglycan compound of formula I are of an unusually low molecular weight and are yet stabilized without derivatization to show excellent dermal penetration to reduce surface wrinkles in skin and exhibit an excellent softening and moisturizing effect as well.
• Egg shell waste produced from the egg processing industry is usually washed with solvents and treated to eliminate unpleasant smells before being used as landfill.
• the calcium carbonate of the shells is only usable upon extensive separation and cleaning procedures which makes the process commercially uneconomical. There is no specific need to pulverize the egg shells within a narrowly bound range either since the processes of the present invention are not dependent on the separation of the inner membrane from the egg shell as in the complex process and equipment described by MacNeil (U.S. Pat. No. 6,176,376) to arrive at pure calcium carbonate.
• Crushed egg shells may be treated with warm water or warm 5% ethanol solution and filtered to remove adhered organic wastes from the surface of the shells.
• the ratio of organic mass to calcium carbonate may be between 1% to 15% w/w. Greater ratios of organic mass would indicate unwashed egg mass present in the crushed egg shells which can lead to the presence of detrimental protein and nucleotide products in the aminoglycan extract.
• the use of egg shell waste as shown herein is unique in the absence of significant antigenic protein and nucleotide components in the extracted media leading to easier methods of extracting purified aminoglycan compound of formula I.
• the egg shells may be additionally pre-treated with ultraviolet light to destroy microbes which may be present even after liquid cleaning.
• the next step comprises treatment of the above egg shell mass to a highly selective extraction of the carbohydrate component in the form of its water-soluble salt.
• the processes involve suspending the egg shell mass in 1:2 to 1:10 volume of solution containing 5% to 40% by weight citrate, glutamate, acetate, pyrrolidonecarbonate, tartrate, glycinate, sulfate, sulfite, nitrate, carbonate and oxalate salts of sodium, potassium, calcium or magnesium or a combination of the above salt solutions as needed. More specifically the mono-valent salts of organic acids are preferred.
• the suspension is held for 1 to 24 hours, more preferably for 6 to 12 hours, with periodic vigorous shaking at temperatures ranging between 10 C and 35 C.
• the suspension is subsequently filtered or centrifuged to remove the aqueous solution containing the appropriate salt of the aminoglycan compounds of formula I.
• the egg shell mass thus separated shows a much looser binding of the membranes to the egg shell and hence may be more easily treated using processes known in the art to separate the pure calcium carbonate containing egg shell from the organic residue.
• the next step comprises the gel precipitation of the aminoglycan in its appropriate salt form from the aqueous solution.
• the processes involve reducing the polarity of the aqueous solution and hence the solubility of the aminoglycan by the sequential addition of any aqueous miscible organic solvent such as alcohols, acetone, dimethylformamide, N-methylpyrrolidinone or 1,4-dioxane.
• the organic solvent is added in lots with mild stirring and cooling to maintain the temperature of the reaction between 20 C to 25 C to yield a white gel formation suspended in the aqueous layer.
• the solution is allowed to stand for 2 to 24 hours until gelation is complete and subsequently filtered or centrifuged to yield a semi-dry extract of aminoglycan compound of formula I. It is important to not allow the extract to be completely dried since a certain amount of the aqueous phase is required during the stabilization process carried out next.
• the final step comprises the stabilization of the low-molecular weight aminoglycan compound of formula I by ordering the molecules in a lipophilic environment to prevent cross-linking which is characteristic of non-acetylated and low-molecular weight aminoglycans as described in the prior art.
• the process involves a sequential addition of two oils whose total weight ratio to the aminoglycan extract is between 1:0.5 and 1:3 of aminoglycan to oils and wherein the individual oil ratios between the two oils are 3:1 to 8:1.
• the first oils should be more hydrophobic in nature and may be oils found typically in plant nuts. Specifically almond and jojoba oils are more preferred as the first oil.
• the second oil component should be more hydrophilic in nature and may be oils typical isolated from herbs and spices from the vegetative parts of plants. Specifically sage, rosemary and lavender oils are more preferred as the second oils.
• the molecular weight of aminoglycan compound of formula I thus isolated is difficult to measure directly and hence I have relied on the measure of intrinsic viscosity to determine the molecular weight.
• the intrinsic viscosities of various solutions containing aminoglycan compound of formula I were found to lie between 4 cm3/gm and 7 cm3/gm and when plotted against standard solutions of hyaluronic acid salts (Mol. Wt. approximately 1.2 MD) led to the assignment of a unique natural ultra low molecular weight for aminoglycan compound of formula I between the range of 15,000 Daltons and 28,000 Daltons.
• An ultra low molecular weight aminoglycan compound of formula I from a natural source has not been previously described in the prior art (for example as summarized by Balazs et al. in U.S. Pat. No. 4,582,865).
• the beaker was placed in a cold water bath and a slow addition of absolute methanol was initiated with slow stirring. After about 200 ml of methanol addition was completed, a cloudy white precipitate began to form and the stirring was stopped. An equal additional quantity of methanol was added slowly and the beaker allowed to stand for 12 hours to make sure gelation was complete. The entire mass was transferred to a filter funnel and filtered to give a cream colored gel of aminoglycan compound of formula I. The precipitate was dried until a moisture content of 5-7% was measured. The final weight of the gel of aminoglycan compound of formula I was 42 grams.
• the gel material containing aminoglycan compound of formula I from Example 1 was mixed with 4 grams of jojoba oil at 15-20 C and stirred vigorously for 20 minutes. The resultant gel was warmed to 25 C and allowed to gently stir for 1 hour. To this mass was added 1 gram of sage oil and the resultant gel was further stirred gently for 10 minutes. The gel is then allowed to slowly cool to 10 C over 4 hours whereby the aminoglycan compound of formula I is ordered into a secondary structure that is stable in the absence of circulating air at room temperature for at least 3 months.
• Example 2 10 grams of the above stabilized gel made as per the procedure shown in Example 2 is added to 50 ml of distilled water containing 3 ml of glycerin and stirred to a uniform suspension. To this suspension is added a melt consisting of 10 grams of emulsifying wax, 10 grams of paraffin wax, 4 grams of white beeswax and 13 grams of a mix of cosmetically useful plant oils such as almond, lavender, sandalwood and walnut and the mixture stirred vigorously to give a uniform cream with excellent physical characteristics and anti-wrinkle properties.
• a melt consisting of 10 grams of emulsifying wax, 10 grams of paraffin wax, 4 grams of white beeswax and 13 grams of a mix of cosmetically useful plant oils such as almond, lavender, sandalwood and walnut and the mixture stirred vigorously to give a uniform cream with excellent physical characteristics and anti-wrinkle properties.
• Ultraviolet spectroscopy has been used to show the absence of potentially antigenic DNA and RNA nucleotides in the aminoglycan compound of formula I extracted herein.
• a 1% solution of the aminoglycan extract from Example 1 in 10% sodium chloride solution was prepared. This solution was subjected to ultraviolet spectroscopy at 257 nanometers to measure the level of nucleotides in the solution. The absence of any absorption at this wavelength was taken as a measure of the absence of nucleotides in the aminoglycan extract from Example 1.
• a small sample of the gel was freeze-dried to give a white solid with a thread like structure which slowly dissolved in water.
• a solution of 1 gram of the powder was made up in 1000 ml of a phosphate buffer at pH 7. Viscosity was determined with an Ostwald viscosimeter at a temperature of 25 C. The relative viscosity of the solution was measured as 0.76 to 0.80. When compared to aminoglycans of known higher molecular weight this viscosity measurement leads to molecular weights for aminoglycan compound of formula I between 15 kD and 28 kD.
• glucosamine in the aminoglycan compound of formula I was determined by the method of Elson and Morgan (Biochem J, Vol. 27, (1933), p. 1894,) on material that had been hydrolyzed for 6 hours with 5N hydrochloric acid at 100 C and evaporated to dryness.
• the glucosamine content of the aminoglycan compound of formula I was between 38% and 41% which matches the expected calculated value.
• the anti-wrinkle properties of the cream produced as per the method described in Example 9 was tested using a 3D imaging system to measure depths of surface wrinkles.
• the method described by S. Jaspers et al, (“Microtopometry Measurement of Human Skin in vivo by a new Digital Optical Projection System”, Preprints 5th Congress of the International Society for Skin Imaging, Wien 1997) was used to show a 25% to 38% reduction in wrinkle depth after 4 weeks of daily use.

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• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
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• Health & Medical Sciences (AREA)
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• Genetics & Genomics (AREA)
• Biotechnology (AREA)
• Polysaccharides And Polysaccharide Derivatives (AREA)
• Cosmetics (AREA)
• Peptides Or Proteins (AREA)
• Extraction Or Liquid Replacement (AREA)
• Processing Of Solid Wastes (AREA)

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• 2006
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