Classifications

• • 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
  • A61Q19/08—Anti-ageing preparations
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
  • A61K8/11—Encapsulated compositions
• • 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
  • 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
  • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
  • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
  • A61K8/9783—Angiosperms [Magnoliophyta]
  • A61K8/9789—Magnoliopsida [dicotyledons]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/16—Emollients or protectives, e.g. against radiation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

• the present invention generally relates to compositions for improving skin condition and appearance, and more particularly to cosmetic compositions including prenylated isoflavonoid compounds effective for inducing a statistically significant upregulation of genes in normal human dermal fibroblasts.
• the present invention also relates to methods for regulating skin condition.
• collagen is a fibrous protein called collagen.
• Collagen especially collagen IAl connects and supports skin tissues. It works hand-in-hand with another fibrous protein, elastin. Basically, collagen provides the skin its form and firmness; elastin gives the same skin elasticity and flexibility.
• the skin produces other fibrous proteins that provide important linkages to the skin cells. For example, fibrillin protein is expressed in the skin and is intimately associated with elastin, surrounding amorphous elastin filaments and providing additional structure and strength.
• U.S. Patent 7351745 discloses a cosmetic composition containing one or more active agents selected from the group consisting of: L-Theanine, S-Methyl-L- Cysteine, S-phenyl-L-Cysteine. According to the patent, the active agents are found effective to increase expression levels of at least one gene selected from the group consisting of collagen 4, collagen 7 and a few other genes.
• U.S. Patent 7,348,034 discloses a cosmetic composition that allegedly works by increasing the synthesis of collagen or elastin and/or preventing or slowing the degradation of collagen and elastin.
• the composition includes grape seed extract, wolfberry extract, and rosehip.
• Flavonoids are a class of plant pigments. They are best known for their antioxidant activities. Certain flavonoids have been used in topical compositions.
• U.S. Patent 6,093,411 discloses that certain selected flavonoid compounds can be used to prevent or treat skin disorders.
• the active agent is selected from isoflavones, coumarins, chromones, dicoumarols, chromanones and chromanols.
• U.S. Publication 2003/0125264 discloses that flavones, flavonols, flavanones, isoflavanones and isoflavones may be used in the treatment of wounds by increasing proliferation of fibroblast cells.
• the present invention relates to a composition having active agents effective to increase collagen IAl and elastin gene expression in normal human dermal fibroblasts.
• the composition comprises a prenylated isoflavonoid and a cosmetically or determatologically acceptable carrier, where the prenylated isoflavonoid is from 0.0000001% to 10%, preferably from 0.00001% to 1%, most preferably from 0.01% to 1% and the carrier is from about 50% to about 99%, preferably from about 75% to about 99%, based on the total weight of the composition.
• the prenylated isoflavonoid is selected from the group consisting of pomiferin, osajin and a combination thereof.
• the present invention relates to a method for regulating skin condition by using a composition containing a safe and effective amount of prenylated isoflavonoid and an acceptable carrier.
• the present invention relates to a topical composition having one or more active agents that improve the appearance and condition of the skin by controlling the extracellular matrix proteins of the skin, especially collagen and elastin.
• the invention is based on the surprising discovery that an appropriate amount of prenylated isoflavoid induces a statistically significant upregulation of collagen IAl and elastin genes in normal human dermal fibroblasts.
• Fibroblasts are cells that grow in the dermal layer of the skin that are responsible for expression of new collagen and elastin into the skin. Such cells can be grown in culture dishes under conditions known as in vitro to examine beneficial influences of topical treatments.
• the prenylated isoflavonoids of the present invention may also act on other cells known to exist in the skin such as, but not limited to, keratinocytes, melanocytes, nerve cells, immune cells, adipocytes and the like.
• the preneylated isoflavoinoids of the present invention may affect cells such as dermal papillae cells, sebocytes, stem cells and the like.
• the prenylated isoflavonoids of the present invention may also act on the stratum corneum of the skin, a portion of the skin which is essentially non-living but which is non-the-less susceptible to the effects of free radicals and solar radiation damage.
• the expression of collagen and elastin can be measured in multiple ways using in vitro assays, but two very practical methods are by human gene microarrays and by Enzyme-Linked Immunosorbent Assays (ELISA).
• the microarray technique employs genomic microchips such as those provided by Affymetrix (Santa Clara, CA) to examine whether a particular treatment influences the fibroblast's genetic predisposition to create collagen or elastin by increasing or decreasing RNA expression.
• the ELISA test examines the actual expression of collagen or elastin by using fluorescently-labeled antibodies specific for the particular protein of interest. Typically, if a treatment upregulates a fibroblast's gene expression of collagen IAl and elastin, then the treatment will likely increase expression of the collagen IAl and elastin proteins as well.
• the present invention provides a composition containing a safe and effective amount of prenylated isoflavonoid(s) and a carrier.
• the composition is effective in stimulating the production of collagen and elastin of the skin when applied to the skin under normal treatment conditions.
• the composition of the present invention is also effective in treating, preventing, and ameliorating the appearance of fine lines, wrinkles, age spots, sagging and other unhealthy skin conditions associated with the loss of collagen and elastin.
• the prenylated isoflavonoids of the present invention is also effective at modulating skin color or tone and for reducing inflammatory responses in the skin.
• the prenylated isoflavonoid is present in an amount of 0.0000001% - 10.0%, preferably in an amount of 0.00001% - 1%, more preferably in an amount of 0.01% - 1%, based on the total weight of the composition.
• compositions of the present invention is a prenylated isoflavonoid of the general structure shown below:
• Prenylated isoflavonoids differ from simple isoflavonoids such as are disclosed, for example, in US 6,093,411 issued to Bissett which have the general structure shown below:
• the prenylated isoflavonoids differ from simple isoflavonoids by containing an additional cylic ring structure, D, and by having an additional terpene moiety attached to ring A that is absent from the simple isoflavonoids of US 6,093,411.
• Prenylated isoflavonoids also differ from simple flavonoids such as are disclosed, for example, in US 2001/0020009 issued to Nair et al., which also lack the additional six-membered ring, D, the attached terpene on ring A and also by having a differing stereochemistry between ring B and ring C than the prenylated isoflavonoids.
• Solvents used for extracting the prenylated isofiaovanoids from Osage orange are known to those skilled in the art and include, but are not limited to, aqueous extraction, aqueous-alcoholic extraction, aqueous-glycol extraction, organic solvent extraction, supercritical carbon dioxide extraction, liquid propane or liquid butane extraction, and the like.
• Methods of extraction can include, but are not limited to, simple solvent extraction, pressurized hot water extraction, steam extraction, counter current extraction, gaseous extraction and the like.
• the extracts may be subjected to further downstream processes including, but not limited to chromatography, vacuum distillation, freeze drying, drum drying, cyclone drying, spray drying, carbon treatment, plate-and-frame filtration, high pressure filtration, centrifugation, and the like.
• the extracts may be further treated to encapsulate them either in lipid encapsulants such as, for example, liposomes, or polymer encapsulat s such as, for example, maltodextrins.
• the extracts may be encapsulated in polymeric matrix, niasome and nanoparticles.
• the naturally sourced material can also further be derivatized (e.g., an ester or ether derivative prepared following extraction from a natural source).
• Flavonoid compounds useful herein are commercially available from commercial sources such as, Gaia Chemical Company (Gaylordsville, CT).
• the prenylated isoflavonoids may also be used for a portion of an additional fermentation process media either in the solid state or as part of a liquid fermentation process with other microorganisms such as, for example, Saccharomyces, Lactobacillus, or E. coli.
• Another essential ingredient of the present invention is a cosmetically or dermatologically-acceptable carrier for prenylated isoflavonoids.
• the carrier is not limited to any particular form.
• the exemplary carriers are water, oil, water-in-oil, oil-in-water, oil-in- water-in-silicon emulsions. These emulsions can cover a broad range of viscosities, e.g, from about 100 cps to about 200,000 cps. These emulsions can also be delivered in the form of sprays using either mechanical pump containers or pressurized aerosol containers using conventional propellants. These carriers can also be delivered in the form of a mousse.
• topical carriers include anhydrous liquid solvents such as oils, alcohols, and silicones (e.g., mineral oil, ethanol, isopropanol, dimethicone, cyclomethicone, and the like); aqueous-based single phase liquid solvents (e.g., hydro- alcoholic solvent systems); and thickened versions of these anhydrous and aqueous-based single phase solvents (e.g., where the viscosity of the solvent has been increased to form a solid or semi-solid by the addition of appropriate gums, resins, waxes, polymers, salts, and the like).
• topical carrier systems useful in the present invention are described in the following four references: "Sun Products Formulary" Cosmetics & Toiletries, vol.
• the carriers of the present invention can comprise from about 50% to about
• compositions of the present invention preferably from about 75% to about 99%, and most preferably from about 85% to about 95%.
• the skin regulating compositions of the present invention may optionally comprise additional skin actives and other components depending on the carrier.
• additional skin actives and other components may be found in U.S. Patent No. 6,093,411.
• Non-limiting examples of such skin actives include vitamin B3 compounds such as those described in PCT application WO 97/39733, published Oct. 30, 1997, to Oblong et al., hydroxy acids such as salicylic acid; exfoliation or desquamatory agents such as zwitterionic surfactants; sunscreens such as 2-ethylhexyl-p-methoxycinnamate, 4,4'-t-butyl methoxydibenzoyl-methane, octocrylene, phenyl benzimidazole sulfonic acid; sun-blocks such as zinc oxide and titanium dioxide; anti-inflammatory agents; anti- oxidants/radical scavengers such as tocopherol and esters thereof; metal chelators, especially iron chelators; retinoids such as retinol, retinyl palmitate, retinyl acetate, retinyl propionate, and retinal; N-acetyl-L-
• Preferred skin actives include hydroxy acids such as salicylic acid, sunscreen, antioxidants and mixtures thereof.
• compositions of the present invention may also be included in the compositions of the present invention.
• a 14 ounce Osage orange fruit was placed into a Waring Juicer and 7 ounces of the liquid contents of the fruit was extracted and removed from the pulp.
• the juice was a viscous, sticky, aqueous mixture which contains the prenylated isoflavonoids osajin and pomiferin along with additional proteins, antioxidants, polysaccharides, acids, alkaloids, vitamins and enzymes common to the plant.
• the extract was further diluted with water and a portion of water insoluble components was removed by fractionation of the two incompatible phases which developed upon dilution.
• the water-soluble components were filtered through diatomaceous earth, followed by filtration through a 0.2 micrometer filter. This formed the basis of an extract of Osage orange containing pomiferin and osajin useful for topical applications.
• Osage orange extract taken from Example 1 was mixed with additional water in a closed container using a magnetic stirring bar at room temperature for 18-24 hours.
• the weight ratio of Osage orange extract to extraction solvent was 1 :4.
• the mixed material was centrifuged at lOOrpm for 15 minutes in a Clay Adams DYNAC centrifuge from Becton Dickinson, and the liquid extract was collected. Alternatively, the mixture can be clarified by filtration.
• the collected liquid extract was diluted in mobile phase solution, filtered through 0.2 ⁇ m syringe filter, and injected for HPLC analysis.
• the aqueous extract created above was further diluted by a factor of 2.
• a 10 ⁇ l of injected material was chromatographed on a Waters XTerra MS Cl 8 column, 4.6x250mm, for 25 minutes under 25°C using isocratic elution at a flow rate of 1.0ml/min with a mobile phase solution constituting 30% A and 70% B, where A is 1% (w/w) acetic acid in water; and B is acetonitrile. All reagents are of HPLC grade. The detection was at 273.5nm.
• Example 3 Testing the influence of Pomiferin using Human Gene Microarrays on normal human dermal fibroblasts.
• the filter was then washed to remove any residual cellular debris from the RNA bound to the glass fibers by subsequently applying 700 ⁇ l of wash solution 1 (1 time) and 500 ⁇ l of wash solution 2 (2 times) to the filter cartridge and centrifuging at 14,000 RPM for 1 minute to pass each wash through the cartridge. After each wash the flowthrough was discarded. After the final wash one final spin was performed without wash solution to remove any residual wash solution in the filter cartridge.
• the RNA bound to the glass fibers within the cartridge was eluted by applying 30 ⁇ l of Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, preheated to 70-80°C) to the cartridge and centrifuging the cartridge in a new collection tube at 14,000 RPM for one minute. The elution process was repeated with an additional 30 ⁇ l of preheated TE buffer. After the RNA was eluted its concentration was quantified using a Ribogreen assay.
• the Ribogreen reagent was provided as a stock solution in DMSO. Prior to use the reagent was diluted 2000 fold in TE buffer. The RNA assay required 200 ⁇ l of diluted Ribogreen reagent per sample to be tested and 1 ml of the reagent for the standards. The diluted reagent was stored protected from light. A series of RNA standards was prepared by diluting purified ribosomal RNA derived from E. coli to the following concentrations: 2 ⁇ g/ml, 1 ⁇ g/ml, 200 ng/ml, 40 ng/ml and 0 ng/ml (blank). Prior to assaying, the RNA samples prepared above were diluted 1000 fold in TE buffer.
• RNA assay 100 ⁇ l of the diluted samples or standards was transferred to the wells of a 96- well plate. The samples and standards were assayed in duplicate. After the samples/standards were added to the plate, lOO ⁇ l of the diluted Ribogreen assay reagent was added to the wells and the plate was gently mixed and allowed to incubate for 5-10 minutes protected from the light. After this incubation the plate was read with a fluorometer using an excitation wavelength of 500 nm and an emission wavelength of 525 nm.
• mRN ⁇ Amplification (Ambion Messa ⁇ eAmp aRNA kit) First Strand cDNA Synthesis: To start the first strand synthesis, 5 ⁇ g of total RNA for each sample was added to 600 ⁇ l PCR tubes and the total volume of liquid in the tube was adjusted to 12 ⁇ l with DEPC H 2 O. To each tube, 1 ⁇ l of T7 Oligo(dT) primer was added and the tube was incubated at 70+2°C for 10 minutes to denature the RNA and then placed on ice to allow the primer to anneal to the poly A ends of the mRNA.
• T7 Oligo(dT) primer was added and the tube was incubated at 70+2°C for 10 minutes to denature the RNA and then placed on ice to allow the primer to anneal to the poly A ends of the mRNA.
• RNAse inhibitor 1 ⁇ l of RNAse inhibitor and 4 ⁇ l of dNTP Mix were added to each tube, and each tube was placed at 42 0 C. 1 ⁇ l of Reverse Transcriptase was added and the tubes were heated and maintained at 42 ⁇ 2°C for 2 hours. At the end of the two hours the tubes were briefly centrifuged to collect all of the fluid at the bottom of the tube and then placed on ice.
• Second Strand Synthesis andcDNA Purification For the synthesis of the second strand of cDNA the following items were added to the tubes above (in this order): 63 ⁇ l DEPC H 2 0, 10 ⁇ l 10 x second strand buffer, 4 ⁇ l dNTP mix, 2 ⁇ l DNA Polymerase and 1 ⁇ l of RNAse H. The tube was mixed and then incubated at 16 ⁇ 2°C for 2 hours. Towards the end of the 2 hour incubation a sufficient quantity of DEPC H 2 O was warmed to 50 ⁇ 2°C and a cDNA purification filter cartridge was equilibrated with 50 ⁇ l of cDNA binding buffer (one cartridge per sample) for at least 5 minutes.
• cDNA binding buffer 250 ⁇ l was added to each tube and thoroughly mixed.
• the contents of the PCR tube was transferred to the cDNA purification filter cartridge.
• the cartridge was placed in a collection tube and centrifuged at 10,000 RPM for 1 minute.
• the flow-through was discarded and 650 ⁇ l of cDNA wash solution was added to the cartridge.
• the cartridge was centrifuged again and the flow-through was discarded, and then centrifuged one last time to ensure that the wash buffer had been completely emptied from the filter.
• the cDNA was eluted by applying 10 ⁇ l of preheated DEPC H 2 O to the filter and centrifuging the filter in a new collection tube at 10,000 RPM for one minute. This elution was performed one additional time to give a total volume of 16-18 ⁇ l of cDNA solution.
• aRNA binding buffer was added to the sample tubes and thoroughly mixed. An additional 250 ⁇ l of absolute ethanol was added to each tube. The mixture was transferred to an aRNA filter cartridge; the cartridge was inserted into a collection tube and centrifuged at 10,000 RPM for 1 minute. The flow-through was discarded and 650 ⁇ l of aRNA wash buffer was added to the cartridge followed by centrifuging at 10,000 RPM for one minute. After discarding the flow through the cartridge was spun one final time to remove all traces of the wash buffer. The cartridge was transferred to a new collection tube and 25 ⁇ l of prewarmed Elution Solution was added to the cartridge.
• the cartridge was incubated for 2 minutes at room temperature and then aRNA was eluted by centrifuging for 1 minute at 10,000 RPM. This elution was performed one additional time to give a total volume of 45-50 ⁇ l of aRNA solution. The final concentration of the aRNA was determined by the Ribogreen assay described above.
• Labeling of aRNA with Fluorescent Dyes (PerkinElmer ASAP RNA Labeling Kit) and Purification of Labeled aRNA [0048] Labeling: Two tubes were prepared for the labeling process, one for Cy3 labeling (green) and one for Cy5 labeling (red). To the Cy3 tube was added 2 ⁇ g of aRNA prepared from the untreated/control sample (the actual color assignment for each sample is not important, but for consistency we normally use Cy3 for the untreated sample) and add enough DEPC H 2 O to bring the total volume up to 4 ⁇ l. To the Cy 5 tube was added 2 ⁇ g of aRNA prepared from the sample treated with the test material and enough DEPC H 2 O to bring the total volume up to 4 ⁇ l.
• a microcon YM-30 filter column was inserted into a collection tube and filled with 400 ⁇ l of TE buffer.
• the Cy3 and Cy 5 probes were combined (12.5 ⁇ l of each) and then added to the microcon filter and thoroughly mixed with the TE buffer.
• the filter was centrifuged at 12,000 RPM for 8 minutes and the flow through was discarded.
• the column was washed twice with 400 ⁇ l of TE buffer, and the flow though was discarded each time. After the final wash the filter column was inverted, placed into a new collection tube and centrifuged at 12,000 RPM for 2 minutes to collect the probe (the probe was further concentrated in a volume of 2-30 ⁇ l of residual TE buffer).
• the hybridization mixture (approximately 110 ⁇ l) was applied to the glass gasket slide and an Agilent Microarray Chip was placed face down on top of this gasket such that the hybridization solution was sandwiched between the glass gasket slide and the microarray face of the chip.
• the top half of the chamber was attached and the connecting thumbscrew was tightened.
• After verifying that there was good bubble formation in the chamber it was placed into the hybridization oven for approximately 17 hours (65°C and rotating at 4 RPM).
• the microarray/glass gasket were removed from the SUREHYB chamber and placed in 50 ml of wash solution 1 (room temperature, 6x SSC, 0.005% Triton X-102).
• the array was transferred to 300 ml of fresh wash solution 1 on a magnetic stir plate. The array was washed while the solution was mixed at medium speed for 10 minutes and then transferred to 300 ml of wash solution 2 (O.lx SSX, 0.005% Triton X-102, 4°C) for 5 minutes. After the final wash the array was centrifuged at 500 RPM for 5 minutes to dry it.
• wash solution 2 O.lx SSX, 0.005% Triton X-102, 4°C
• microarrays were scanned with an Axon GenePix 410OA Scanner with the scanning resolution set to 10 ⁇ m and analyzed with GenePix Pro software. During the initial scan the PMT gains for the scanner were adjusted such that the cy5/cy3 image count ratios are between 0.88 and 1.12.
• the level of gene expression is related to the fluorescence intensity of the probed gene marker on the microarray. Since it is possible to have differences in labeling efficiency when making the Cy3 and Cy5 probes it is essential to normalize the fluorescence measurements between the two respective dyes before looking at changes in gene expression. Fluorescence intensities for the microarrays were subjected to global normalization. The total fluorescent signal for both dyes was normalized with a correction factor that made the ratio of total intensities for both dyes equal to one.
• the ratio of Cy3/Cy5 (untreated/treated) fluorescence intensity is greater than 1.3 or less than 0.7. This relates to a change in gene expression of at least +/- 30%
• the fluorescence intensity of the gene marker is greater than the background intensity.
• the gene feature is clearly marked specifically by the aRNA probes and is not due to non-specific fluorescence (i.e. SDS streaks will leave fluorescent trails).
• the first two criteria can be filtered via computer analysis of the data.
• the last criterion requires a visual inspection of the array spot to confirm.
• Cy3/Cy5 would be equivalent to F635/F532 and the fluorescence ratio is identified as the "Ratio of Medians 635/532".
• the IDl and Name identify the gene and its location on the human genome.
• Table 1 Gene expression for Type IAl Collagen results from treatment of normal human dermal fibroblasts with 0.05% Pomiferin.
• Example 4 Examination of pomiferin's influence on collagen IAl and elastin protein expression using ELISA assays.
• Untreated cells just received DMEM with 1.5% FBS. The cells were incubated for 48 hours and at the end of the incubation period cell culture medium were collected and either stored frozen (- 75 0 C) or assayed immediately. Materials were tested in triplicate.
• the cell culture medium was removed (see above) and the fibroblasts were washed twice with PBS to remove any remaining test material.
• 1 ml of media supplemented with 0.5 mg/ml MTT was added to each well and the cells were incubated for 1 hour at 37+2 0 C and 5+1% CO 2 .
• the MTT solution was removed and the cells were washed again once with PBS and then 1 ml of isopropyl alcohol was added to the well to extract the purple formazin crystals. Two hundred microliters of the isopropyl extracts was transferred to a 96-well plate and the plate was read at 540 run using isopropyl alcohol as a blank.
• a series of type I C-peptide standards was prepared ranging from 40 ng/ml to 640 ng/ml.
• An ELISA microplate was prepared by removing any unneeded strips from the plate frame.
• 100 ⁇ l of peroxidase-labeled anti procollagen type I-C peptide was added, followed by 20 ⁇ l of either sample or standard.
• the microplate was covered and allowed to incubate for 3 + 0.25 hours at 37°C. After the incubation each well was washed three times with 400 ⁇ l of wash buffer.
• Soluble ⁇ -elastin was dissolved in 0.1 M sodium carbonate (pH 9.0) at a concentration of 1.25 ⁇ g/ml. 150 ⁇ l of this solution was applied to the wells of a 96-well maxisorp Nunc plate and the plate was incubated overnight at 4°C. On the following day the wells were saturated with PBS containing 0.25% BSA and 0.05% Tween 20. The plate was incubated with this blocking solution for 1 hour at 37 0 C and then washed two times with PBS containing 0.05% Tween 20.
• a set of ⁇ -elastin standards was generated ranging from 0 to 100 ng/ml.
• the mean MTT absorbance value for the negative control cells were calculated and used to represent 100% value for cell number.
• the individual MTT values from the cells undergoing the various treatments were divided by the mean value for the negative control cells and expressed as a percent to determine the change in cell number caused by each treatment. If the MTT assay demonstrated a reduction in cell viability of greater then 10%, the test was considered invalid. In the assay with pomiferin, the MTT assay demonstrated cell survivals of 100% at all concentrations tested Procollagen and Elastin Concentrations
• PVDF membrane was prewet in methanol, equilibrated with TBS (TBS:
• the membrane was removed from the Bio-Dot apparatus, washed in TBS for 5-10 minutes and then placed into blocking solution (TBS with 1% non-fat milk powder) and allowed to incubate for at least 1 hour at room temperature on a rocking platform.
• TBS blocking solution
• the membrane was transferred to 20 ml of TBST (TBS with
• Imager FX and scanned using an excitation laser and emission filter combination appropriate for the fluorophore. Images produced by the scanner were then analyzed using ImageJ image analysis software.
• the mean MTT absorbance value for the negative control cells was calculated and used to represent 100% cell viability.
• the individual MTT values from the cells undergoing the various treatments were then divided by the mean value for the negative control cells and expressed as a percent to determine the change in cell viability caused by each treatment.
• RFU Fluorescence Units
• Phase B Phase B to Phase A. Mix for 20 minutes.
• Phase C Phase C and mix until uniform. Turn off the heat.
• Phase B ingredients in order shown, thoroughly mixing each component until homogeneous before adding the next ingredients. 3. Slowly add Phase A to Phase B with good mixing. Gradually increase agitation to high shear as mixture thickens. Continue agitation for 10 minutes.
• Example 1 The Osage orange from Example 1 was encapsulated into a polymeric matrix using the techniques outlined in US Patent Publication No. 2003/0198682 Al.
• Example 1 The Osage orange extract of Example 1 was formulated into an aqueous alcoholic tonic using the following formulation and process:
• Example 1 The osage orange extract of Example 1 was formulated into a body wash using the following formulation and process.
• Cocamide MEA Velvetex BA-35, and mix until uniform.
• the osage orange extract from Example 1 was included as part of a fermentation media containing the Yeast Saccharomyces cerevisiae.
• a sample of the extract from Example 1 was placed into an aqueous mixture of Baker's Yeast growth media obtained from Red Star Yeast (Milwaukee, WI).
• the media was inoculated with an active Saccharomyces cerevisiae yeast culture also obtained from Red Star and the mixture was allowed to ferment under controlled aerobic conditions to provide a Live Yeast Cell Derivative (LYCD) obtained using stress conditions as described in US patent 2,239,345.
• LYCD Live Yeast Cell Derivative
• This example illustrates a sub-micron emulsion concentrate that contains an osage orange extract prepared as described in Example 1.
• Ingredient Wt% Ingredient Wt%

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• General Health & Medical Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• Epidemiology (AREA)
• Birds (AREA)
• Dermatology (AREA)
• Engineering & Computer Science (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Medicinal Chemistry (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Pharmacology & Pharmacy (AREA)
• Biotechnology (AREA)
• Botany (AREA)
• Microbiology (AREA)
• Mycology (AREA)
• Gerontology & Geriatric Medicine (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Toxicology (AREA)
• Rheumatology (AREA)
• Pain & Pain Management (AREA)
• Cosmetics (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Medicines Containing Plant Substances (AREA)

Priority Applications (2)

Applications Claiming Priority (4)

Publications (1)

Family

ID=41610737

Family Applications (1)

Country Status (4)

Cited By (1)

Families Citing this family (8)

Citations (2)

Family Cites Families (16)

• 2009
  • 2009-07-30 US US12/512,368 patent/US9000033B2/en not_active Expired - Fee Related
  • 2009-07-31 JP JP2011521342A patent/JP6223657B2/ja not_active Expired - Fee Related
  • 2009-07-31 EP EP09803624.7A patent/EP2303010B1/en not_active Not-in-force
  • 2009-07-31 WO PCT/US2009/052325 patent/WO2010014861A1/en not_active Ceased
• 2015
  • 2015-10-30 JP JP2015214135A patent/JP6257052B2/ja not_active Expired - Fee Related

Patent Citations (2)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events