Patent Application: US-3598708-A

Abstract:
the present invention relates to vitamin c preparations which enhance absorption of vitamin c into cells , and prolong the retention of vitamin c within the blood plasma and tissue of mammals , such as humans . the vitamin c preparations of the present invention include lipophilic molecules which improve the absorption of vitamin c resulting in higher plasma and cellular levels .

Description:
the term “ vitamin c ,” unless otherwise stated , refers to ascorbic acid and pharmaceutically acceptable salts thereof , including , but not limited to , mineral salts of ascorbic acid , effervescent vitamin c ( e . g ., a combination of ascorbic acid , citric acid and sodium bicarbonate ), chelates of ascorbic acid , and alkaline salts of ascorbic acid . suitable lipophilic molecules include , but are not limited to , those derived from natural waxes such as , but is not limited to , sugar cane wax , rice bran wax , carnauba wax , candelilla wax , japan wax , ouricury wax , bayberry wax , shellac wax , sunflower wax , orange wax , and beeswax . according to one preferred embodiment , the lipophilic molecules are derived from rice bran wax , carauba wax , cadelilla wax , and beeswax . according to a more preferred embodiment , the lipophilic molecules are derived from rice bran wax . suitable lipophilic molecules extracted from natural waxes include , but are not limited to , palmitic acid , linoleic acid , linolenic acid , oleic acid , and steric acid . according to one preferred embodiment , the vitamin c preparation includes one or more or all of the following lipophilic molecules at the recited weight ratios : in one embodiment , the vitamin c preparation includes one or more or all of the following lipophilic molecules at the recited weight percentages : about 5 . 0 - 15 . 0 % tetratriacontanol , based upon 100 % total weight of the lipophilic molecules in the vitamin c preparation . the mixture of lipophilic molecules can be obtained by ( 1 ) saponification of a wax ( e . g ., a natural wax ), ( 2 ) solidifying and grinding the saponified wax to a d 90 less than 2000 microns ( e . g ., 100 - 500 microns or 500 - 2000 microns ), ( 3 ) extracting the ground material with acetone or an alcohol ( e . g ., ethanol or isopropanol ), and ( 4 ) optionally solidifying and grinding the extracted material to a d 90 less than 2000 microns ( e . g ., 100 - 500 microns or 500 - 2000 microns ). the natural waxes undergo saponification or hydrolysis before the extraction procedure . for saponification , the natural waxes are heated using a jacketed kettle at 90 ° c . for 3 hours until the wax was completely melted , koh is added , and the mixture is held at 90 ° c . for 1 hour with stirring . for hydrolysis , the natural waxes are heated using a jacketed kettle at 90 ° c . for 3 hours until the wax was completely melted , sulfuric acid aqueous solution is added , and mixture is held at 90 ° c . for 1 hour with stirring . after 1 hour of stirring the saponified or hydrolyzed wax is poured into cart trays and dried at 21 . 1 ° c . before undergoing the extraction procedure . the extraction of the natural waxes may be performed by either dispersed - solids extraction or immersion type percolation extraction . for the dispersed - solids extraction , the natural waxes are ground to a particle mesh size of 100 to 425 microns and subjected to liquid extraction in a dispersed - solids extraction system . in the case of immersion type percolation extraction , the natural waxes are ground to a particle mesh size of 500 to 2000 microns and subjected to liquid extraction in a solid - liquid immersion type percolating extractor system . in both types of extraction equipment , the natural mixture of aliphatic alcohols , saturated fatty acids , and omega - 3 , omega - 6 , omega - 9 fatty acids is selectively extracted with adequate hot organic solvents such as acetone and ethanol with a temperature range of 55 ° c . to 75 ° c . the extractions are purified with hot organic solvents such as hexane , heptane , and acetone ; recovered ; and dried . the extractions contain a mixture of aliphatic alcohols having 17 to 34 carbon atoms , saturated fatty acids having 16 to 30 saturated carbon atoms , and omega - 3 , omega - 6 , omega - 9 fatty acids with melting point between 70 and 80 ° c . the ratio of the natural wax particles to hot liquid extractants is from 1 to 4 and 1 to 10 . according to one preferred embodiment , the extractions with hot organic solvents at 60 ° c ., and the ratio of natural wax particles to hot liquid extractants is 1 to 8 . suitable bioflavonoids include , but are not limited to , rutin , naringin , hesperidin , neohesperidin , neohesperidin dihydrochalcone , naringenin , hersperitin , nomilin , and gallic acid . according to one preferred embodiment , the vitamin c preparation contains hesperidin , gallic acid , and optionally other bioflavonoids . according to one preferred embodiment , the vitamin c preparation includes one or more or all of the following bioflavonoids at the recited weight ratios : in one embodiment , the vitamin c preparation includes one or more or all of the following bioflavonoids at the recited weight percentages : according to one embodiment , the vitamin c preparation includes vitamin c and the lipophilic molecules at a weight ratio ranging from about 1000 : 1 to about 10 : 1 . according to a preferred embodiment , the weight ratio ranges from about 100 : 1 to about 8 : 1 . according to a preferred embodiment , the vitamin c preparation includes at least about 90 % by weight of vitamin c and about 0 . 1 % by weight of the lipophilic molecules based upon 100 % total weight of the vitamin c preparation . more preferably , the vitamin c preparation includes from about 90 to about 99 % by weight of vitamin c and from about 1 to about 8 % by weight of lipophilic molecules . according to another embodiment , the vitamin c preparation includes from about 90 to about 98 % by weight of vitamin c and from about 2 to about 7 % ( e . g ., about 5 %) by weight of lipophilic molecules . according to a preferred embodiment , the vitamin c preparation includes at least about 90 % by weight of vitamin c , from about 0 . 1 to about 9 % by weight of lipophilic molecules , and from about 0 . 1 to about 5 % by weight of bioflavonoids . according to another embodiment , the vitamin c preparation includes about 200 to 40 , 000 iu vitamin c . the vitamin c preparation is preferably in the form of an oral dosage form , such as beads , pellets , granules , capsules ( soft or hard ), sachets , tablets , powders , dispersible powders capable of effervescing upon addition of water , aqueous or oily suspensions , emulsions , syrups , elixirs , or lozenges . for example , the oral dosage form can be an chewable tablet or gum , oral liquid dosage form , such as a suspension in an aqueous or non - aqueous liquid solution , or an emulsion which can be a soft drink , tea , milk , coffee , juice , sports drink , or water . the vitamin c preparation can also be incorporated into various products , such as nutritional supplements ( including vitamins and multi - vitamins ), foods ( including health food products such as nutrition bars ), and drinks ( including fruit juices such as energy drinks ). generally , the daily dosage of the vitamin c preparation on a vitamin c weight basis can range from 30 mg to 2 g . for instance , the daily dosage can be 60 mg to 1 g or 60 mg to 500 mg . desirably , the daily dosage ranges from 60 mg to 500 mg ( e . g ., the daily dosage can be 400 mg ). according to one preferred embodiment , the daily dosage ranges from 60 mg to 200 mg ( e . g ., the daily dosage can be 60 , 100 , or 200 mg ). the daily dose can be achieved by administration of a single dosage form of the invention or alternatively , two or more such dosage forms . preferably , the daily dose is achieved by administration of only one or two dosage forms ( e . g ., once daily dosing or b . i . d .). therefore , the present invention includes , but is not limited to , dosage forms containing 30 , 60 , 100 , 200 , 400 , 500 , or 1000 mg of the vitamin c preparation ( on a vitamin c weight basis ). the vitamin c preparation may include one or more excipients or additives . suitable excipients and additives include , but are not limited to , additional antioxidants ( e . g ., phenolic compounds ), inert diluents ( such as lactose , sodium carbonate , calcium phosphate , and calcium carbonates ), granulating and disintegrating agents ( such as corn starch and algenic acid ), binders ( such as starch ), lubricants ( such as magnesium stearate , stearic acid and talc ), preservatives ( such as ethyl or propyl p - hydroxybenzoate ), colorants , flavoring agents , release modifying agents , thickeners , and any combination of any of the foregoing . suitable antioxidants include , but are not limited to , bioflavonoids , flavonoids , flavonols , flavanones , flavones , flavonals , flavanolols , and flavanols . suitable inert solid diluents include , but are not limited to , calcium carbonate , calcium phosphate and kaolin . suitable diluents for soft capsules include , but are not limited to , water and oils such as peanut oil , liquid paraffin , corn oil , wheat germ oil , soybean oil , and olive oil . aqueous suspensions or dispersions contain the vitamin c preparation , for example , in fine powder form together with one or more suspension or dispersion ( or wetting ) agents . suitable suspension agents include , but are not limited to , sodium carboxymethylcellulose , methylcellulose , hydroxypropylmethylcellulose , sodium alginate , polyvinyl - pyrrolidone , gum tragacanth and gum acacia . suitable dispersing or wetting agents include , but are not limited to , lecithin , condensation products of an alkylene oxide with fatty acids , condensation products of ethylene oxide with long chain aliphatic alcohols , condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate , or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides . dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water contain the vitamin c preparation , for example , together with a dispersing agent , wetting agent , or suspending agent . suitable dispersing agents , wetting agents , and suspending agents include those mentioned above . oily suspensions may be formulated by suspending the vitamin c preparation in an oil , such as an vegetable oil or a mineral oil . the oily suspensions may also contain a thickening agent such as carnauba wax , candelilla wax , rice bran wax , beeswax , hard paraffin , or cetyl alcohol . the vitamin c preparation may be in the form of an oil - in - water emulsion . the oily phase may be a vegetable based oil or a mineral based oil . suitable emulsifying agents include , for example , naturally occurring gums such as acacia and tragacanth gum , naturally occurring phosphatides such as soy bean , lecithin , esters and partial esters derived from fatty acids and hexitol anhydrides and condensation products of partial esters with ethylene oxide , such as polyoxyethylene sorbitan monooleate . syrups and elixirs may be formulated with sweetening agents such as glycerol , propylene glycol , sorbitol , aspartame , or sucrose , and may also contain a demulcent , preservative , flavoring , or coloring agent . the vitamin c preparation may be also in a form suitable for administration by inhalation ( e . g ., as a finely divided powder or a liquid aerosol ), or for parenteral administration ( e . g ., as a sterile aqueous or oily solution for intravenous , subcutaneous , intramuscular dosing or as a suppository for rectal dosing ). administration of the vitamin c preparation by these non - oral routes avoids gastrointestinal side effects , which may accompany high doses of vitamin c released in the stomach . the vitamin c preparation can also be delivered topically , for example , to protect the skin from free radicals , promote wound healing ( for instance , for healing cuts , abrasions , sun damage ( e . g ., sun burn ), wrinkles , and scars ), and / or reduce inflammation . the vitamin c preparation of the invention provides superior penetration of vitamin c through the skin than vitamin c alone . transdermal delivery of the vitamin c preparation permits systemic delivery of the vitamin c while avoiding gastrointestinal side effects . the topical formulation containing the vitamin c preparation can be in the form of a solution , suspension , lotion , emulsion , ointment , cream , or gel . according to a preferred embodiment , the topical formulation is a cream or lotion . the formulation may include additional active ingredients . these formulations may prepared by methods known in the art , and typically include a topically acceptable vehicle . one embodiment is a topical formulation containing about 0 . 5 to about 25 % by weight of the vitamin c preparation of the present invention , based upon 100 % total weight of the topical formulation . for instance , the topical formulation can contain 0 . 5 - 2 %, 1 - 2 %, 1 - 5 %, 1 - 10 %, 5 - 15 %, 5 - 20 %, or 10 - 20 % by weight of the vitamin c preparation . the vitamin c preparation could be used to coat a medical device that is then positioned to a desired target location within the body , whereupon the vitamin c preparation elutes from the medical device . preferably , the coating includes a therapeutically effective amount of the vitamin c preparation . in one embodiment , the medical device is positioned so that the vitamin c preparation is released in a therapeutically effective amount to a targeted site such as a diseased or injured tissue or organ . the device can be introduced temporarily or permanently into a mammal ( e . g ., a human ) for the prophylaxis or therapy of a medical condition , or to augment the immune system . the device can be introduced subcutaneously , percutaneously , or surgically . the medical device can be selected from stents , synthetic grafts , artificial heart valves , artificial hearts and fixtures to connect the prosthetic organ to the vasculature , venous valves , abdominal aortic aneurysm grafts , inferior venal caval filters , catheters including permanent drug infusing catheters , embolic coils , embolic materials used in vascular embolization mesh repair materials , a dracon vascular particle orthopedic metallic plates , rods , screws , and vascular sutures . the vitamin c preparation may be formulated to provide immediate release or controlled release ( e . g ., sustained release ) of the vitamin c preparation , for example , to provide effective doses of vitamin c over extended periods of time to prolong the biological activity and beneficial biochemical functions of vitamin c . one embodiment of the invention is a controlled release dosage form ( such as a solid dosage form ) containing about 200 to 40 , 000 iu vitamin c , about 1 to 100 mg of lipophilic molecules , and 1 to 500 mg of bioflavanoids . for example , the controlled release dosage form may release about 10 to about 35 % by weight of the total vitamin c preparation within about 2 hours in an in vitro dissolution test , and about 40 to about 70 % by weight of the total vitamin c preparation within about 8 hours . according to another embodiment , the controlled release dosage form may release about 50 % by weight of the total vitamin c preparation within about 2 hours in an in vitro dissolution test , and more than 90 % by weight of the total vitamin c preparation within about 6 or 8 hours . any type of controlled release system known in the art can be used . the in vitro dissolution test is conducted using the basket method ( apparatus 1 ) with 900 ml 0 . 1n hcl as the medium run at 100 rpm at a temperature of 37 ° c . the samples are filtered through whatman filter paper # 1 and the amount of vitamin c is calculated based on the equivalence to standard dicholorophenol - indophenol solutions . solid controlled release dosage forms ( e . g ., tablets ) can be formulated ( e . g ., coated ) so as to prolong the release of the vitamin c preparation into the gastrointestinal tract , or to prevent the release of the vitamin c preparation in the stomach in order to prevent or attenuate the gastrointestinal side effects which can accompany high doses of vitamin c released in the stomach . for example , the vitamin c preparation can be enteric coated so as to prevent significant release of the preparation in the stomach . controlled release of the vitamin c preparation can prolong therapeutic and / or immunoprotective systemic concentrations of vitamin c in a person . one embodiment of the invention is a three layer controlled release dosage form ( e . g ., a tablet ) where each layer contains a vitamin c preparation of the invention . the vitamin c preparation of each layer can be the same or different . at least one of the layers provides controlled release of the vitamin c preparation . for example , the dosage form can include ( i ) a first layer , ( ii ) a second layer , and ( ii ) an outer layer surrounding the first and second layers , where the first layer and outer layer provide controlled release of the vitamin c preparation ( s ) and the second layer provides immediate release of the vitamin c preparation . according to one preferred embodiment , the outer layer releases substantially all (& gt ; 90 %) of the vitamin c preparation in a controlled manner within 60 , desirably 30 , and even more desirably 20 minutes , as determined by the aforementioned in vitro dissolution test . the second layer provides immediate release of the vitamin c preparation contained therein . finally , the first layer releases the vitamin c preparation contained therein in a controlled manner over at least 6 hours ( e . g ., substantially of the vitamin c preparation may be released within 6 - 10 hours or 6 - 8 hours ), as determined by the aforementioned in vitro dissolution test . transdermal patch devices can also provide controlled administration ( e . g ., continuous or other sustained administration ) of the vitamin c preparation . methods for preparing controlled release transdermal formulations are known in the art . for example , the transdermal device may contain an impermeable backing layer which defines the outer surface of the device and a permeable skin attaching membrane , such as an adhesive layer , sealed to the outer layer in such a way as to create a reservoir between them wherein the therapeutic agent is placed ( e . g ., a bandage or patch ( including a time released patch )). other suitable controlled release systems include , but are not limited to , long - term sustained implants , aqueous or oily suspensions , emulsions , syrups , elixirs , or lozenges , chewable tablet or gum , foods , beverages , osmotic systems , and dissolution system ( e . g ., effervescent oral dosage form ). the vitamin c preparation of the present invention is preferably administered orally to a mammal ( e . g ., a human ), but it can also be administered by other routes of administration , such as intravenously or subcutaneously . the vitamin c preparation of the present invention may be prepared by methods well known in the art , such as mixing the vitamin c , lipophilic molecules , optionally bioflavanoids , and any desired excipients . saponification : 25 kg of rice bran wax was heated using a jacketed kettle at 90 ° c . for 3 hours until the wax was completely melted . 4 . 67 l of 8 . 0 m koh ( 450 g / l ) in water was slowly added with continuous stirring and heating . the mixture was held at 90 ° c . for 1 hour with stirring . after 1 hour the saponified wax was poured into cart trays and dried at 21 . 1 ° c . the 32 . 1 kg of cooled dried saponified wax was then ground to a powder ( 100 - 425 or 500 - 2000 microns ). extraction : 9 . 6 kg of the saponified wax was placed in 8 extraction thimbles ( 1 . 2 kg of saponified wax per extraction thimble ). 100 l of acetone were pumped into a 200 l cylindrical - bottom flask and connected to a soxhlet system . the system was refluxed for approximately 24 hours , and the extract was pumped to a jacketed reactor . the extract was chilled to approximately 10 ° c . with 20 rpm agitation ( 20 rpm ) for 10 hours . the chilled extract was then centrifuged in a vertical basket centrifuge . the collected solid was poured into trays and vacuum dried for 16 hours . the dried solid was then ground to a powder . a jacketed mixer was charged with dry powder of 58 kg of vitamin c , 0 . 75 kg of the lipid metabolites prepared above and 1 . 5 kg of bioflavonoids ( anmar international ltd ; bridgeport , conn .). the mixer was then turned on ( agitation is initiated — plows ) to create a homogenous mixture of dry powder . the high speed shearing devices ( choppers ) were initiated for 1 minute . hot water was then pumped through the jacket of the mixer to heat the mixture to 80 ° c . with continuous mixing ( plows only ) for 15 minutes for complete encapsulation . the encapsulated mixture was cooled by running chilled water ( 10 ° c .) through the jacket under continuous mixing for 1 hour until a free - flowing powder was formed . the powder was discharged into a double polyethylene - lined container and then passed through a comminuting mill running at approximately 2500 rpm equipped with a 0 . 15 mm screen . the milled powder was collected into appropriately labeled , double polyethylene - lined drums and reconciled . cells from the human t - lymphoblastic h9 cell line were starved of vitamin c for 18 hours in serum - free media and subsequently suspended in 50 μm of ( 1 ) ascorbic acid ( aa ), ( 2 ) calcium ascorbate ( caa ), ( 3 ) calcium ascorbate - calcium threonate - dehydroascorbate ( commercially available as ester - c ® from nature &# 39 ; s value of coram , n . y .) ( ester - c ®), or ( 4 ) the vitamin c preparation of example 1 ( pwc ). at the times indicated in fig1 , cells were harvested and measured for vitamin c and protein content . the cellular vitamin c levels of the cells were measured using the 2 , 4 - dinitrophenylhydrazine spectrophotometric technique ( bessey et al ., 1947 ). over a two hour period , the level of vitamin c uptake from example 1 was consistently higher than that observed with ascorbic acid , calcium ascorbate , and calcium ascorbate - calcium threonate - dehydroascorbate ( see fig1 ). at fifteen minutes , cellular vitamin c levels ranged from 7 ± 1 . 4 nmol / mg cellular protein with ascorbic acid , to over double that amount ( 15 ± 2 . 4 nmol / mg protein ) with the vitamin c preparation of example 1 . the absorbed vitamin c levels rose significantly with time , peaking at approximately two hours with cellular levels ranging from 31 nmol / mg protein for ascorbic acid and 50 nmol / mg protein for the vitamin c preparation of example 1 . in order for vitamin c to exert its beneficial effects , it must be taken up into the cell . to date , vitamin - c lipid metabolites exhibits the greatest amount of vitamin c uptake and retention as compared to all other vitamin c formulations . the ability to inhibit pesticide - induced t - lymphocyte aggregation was determined for the formulation of example 1 and other vitamin c formulations . the human t - lymphoblastic h9 cell line was incubated with vehicle (-) or with one of two activators of t - lymphocyte aggregation , phytohemagglutinin ( pha ; 10 μm ) or bifenthrin ( 10 mm ). the cells were immediately treated with 0 . 5 μm of ( 1 ) ascorbic acid ( aa ), ( 2 ) calcium ascorbate ( caa ), ( 3 ) calcium ascorbate - calcium threonate - dehydroascorbate ( ester - c ®), or ( 4 ) the vitamin c preparation of example 1 ( pwc ) for 30 minutes at 37 ° c . after treatment , the ability of each formulation to inhibit homotypic aggregation was measured by counting aggregate size at 400 × magnification . the vitamin c preparation of example 1 , inhibited the aggregation of the t - lymphocytes induced by the pesticide pha or the pesticide bifenthrin by 88 % and 84 % respectively ( table 2 ). the reduction in t - lymphocyte aggregation was greater following treatment with the vitamin c preparation of example 1 than any of the other formulations . leukocyte cell - cell adhesion is associated with xenobiotic induced hyperactivation and inflammatory damage , and vitamin c has been shown to prevent cigarette smoke - induced leukocyte aggregation and attachment to vascular endothelium ( lehr et al ., 1994 ; weber et al ., 1996 ). as shown in table 2 , vitamin c has also been shown to reduce pesticide mediated t - cell hyperactivation . given that the formulation of the current invention has greater ability to prevent pesticide - induced t - cell aggregation than other vitamin c formulations , suggests that the formulation of the present invention will provide greater protection against other deleterious xenobiotics . briefly , 200 ml of a 1 , 2 . 5 , 5 , 10 , or 20 μg / ml solution of the vitamin c preparation of example 1 was mixed with 50 μl of a 659 μm 1 , 1 - diphenyl - 2 - picryl hydrazyl ( dpph ) solution and incubated at 25 ° c . for 20 minutes . free radical scavenging activity of the vitamin c preparation of example 1 was measured by the reduction of 1 , 1 - diphenyl - 2 - picryl hydrazyl ( dpph ) to 1 , 1 - diphenyl - 2 - picryl hydrazine at an absorbance of 510 nm . the results are shown in fig2 . the vitamin c preparation dose dependently scavenged dpph free radicals . the vitamin c preparation demonstrated excellent scavenging ability by reducing the dpph - induced free radical concentration by 93 % at its maximum concentration . the peroxyl radical oxygen reactive species ( orac ) scavenging ability of the vitamin c preparation was also determined . the orac assay detects free radical damage to fluorescein induced by 2 , 2 ″- asobix dihydrochloride ( aaph ; 153 mm ), and the change is measured by fluorescence spectrophotometry . antioxidants inhibit the free radical range damage to the fluorescent compound and prevent the reduction in fluorescence . the results are shown in table 3 . the results from different concentrations of the vitamin c preparation of example 1 were compared to the known antioxidant trolox ®. the orac results are expressed as trolox ® equivalents ( 6 - hydroxy - 2 , 5 , 7 , 8 - tetramethylchroman - 2 - carboxylic acid ; te ) per gram of sample . vitamin c is a chemical reducing agent for many intracellular and extracellular reactions such as oxidative dna or protein damage , low - density lipoprotein oxidation , lipid peroxidation , oxidants , the formation of nitrosamines in gastric juice , extracellular oxidants from neutophils , and endothelium dependent vasodilation . the vitamin c preparation of the present invention , which exhibits potent antioxidant and free radical scavenging effects in vitro , can serve as a good vitamin c preparation to prevent such damage thus contributing to the protection against cancer , cardiovascular diseases , atherosclerosis , and other age - related diseases caused by cytotoxic , genotoxic , and proinflammatory mechanisms . pc12 cells were treated with 100 ng / ml of nerve growth factor ( ngf ) and incubated for a 24 hour period followed by treatment with either vehicle (-) or various 50 μm of ( 1 ) ascorbic acid ( aa ), ( 2 ) calcium ascorbate ( caa ), ( 3 ) calcium ascorbate - calcium threonate - dehydroascorbate ( ester - c ®), or ( 4 ) the vitamin c preparation of example 1 ( pwc ). the formation of neurites were measured at hours 1 , 3 , 6 , 9 , 12 , and 24 . the results are shown in fig3 . pc12 cells responded to ngf treatment by extending neurites . the vitamin c preparation of example 1 significantly enhanced the ngf - induced neurite outgrowth in 12 % of the cells by the first hour . in fact , the vitamin c preparation was the only formulation that resulted in a significant augmentation of ngf - induced neurite outgrowth , suggesting that this is the only formulation that would aid in protection against neurodegenerative diseases . the ability to promote fibroblast adhesion to fibronectin was determined for the formulation of example 1 and other vitamin c formulations . nih3t3 fibroblastoma cells were seeded onto fibronectin coated plates pretreated with either vehicle (-) or various 50 mm of ( 1 ) ascorbic acid ( aa ), ( 2 ) calcium ascorbate ( caa ), ( 3 ) calcium ascorbate - calcium threonate - dehydroascorbate ( ester - c ®), or ( 4 ) the vitamin c preparation of example 1 ( pwc ). the plates were incubated for 15 minutes at 37 ° c . the unattached cells were removed by aspiration and the attached cells were fixed , stained , and counted in triplicate . results are shown in fig4 . the vitamin c preparation of example 1 enhanced fibroblast adhesion to fibronectin by over three - fold . in addition to adhesion , fibroblast spreading on fibronectin is an important next step to migration and wound healing performance . the human serum vitamin c , plasma c - reactive protein , oxidized ldl , and urine uric and oxalate levels were determined for the formulation of example 1 and other vitamin c formulations . healthy volunteers maintained a low vitamin c diet for 14 days . following an overnight fast , volunteers received a single oral dose of 1000 mg or either ( 1 ) ascorbic acid ( aa ), ( 2 ) calcium ascorbate ( caa ), ( 3 ) calcium ascorbate - calcium threonate - dehydroascorbate ( commercially available as ester - c ® from nature &# 39 ; s value of coram , n . y .) ( ester - c ®), or ( 4 ) the vitamin c preparation of example 1 ( pwc ). blood samples were collected immediately prior to the oral dose administration and at various time points post ingestion . urine was collected over a 24 - hour time period and saved for oxalate and uric acid assays . serum vitamin c levels were measured by hplc with coulometric electrochemical detection . plasma c - reactive protein and oxidized ldl were measured by enzyme linked immunosorbent assay ( elisa ) and urine uric acid and oxalate levels were measured by enzymatic methods . the vitamin c preparation of example 1 is more rapidly absorbed and leads to higher serum vitamin c levels and greater reduction of plasma levels of inflammatory and oxidative stress markers than other forms of vitamin c . * statistically significant deference compared to calcium ascorbate . at one hour p = 0 . 0026 for pwc and p = 0 . 049 for ester - c . at two hours , p = 0 . 0009 . at four hours p = 0 . 0278 for pwc and 0 . 0477 for ester c . at six hours , p = 0 . 0470 ** statistically significant difference from ascorbic acid ( p = 0 . 045 ). note that the reductions in oxldl were not significantly different for any vitamin c supplementation with a before - and - after comparison ; however , the drop observed with pwc was significantly greater than the drop observed with ascorbic acid . all statistically significant differences are noted . data are presented as the mean + s . e . m . all 0 time points were immediately prior to oral administration of the vitamin c formulation bessey o , lowry o , brock m : the quantitative determination of ascorbic acid in small amount of white blood cells and platelets . jbc 1947 , 168 ( 1 ): 197 - 205 boothby l a , doering p l : vitamin c and vitamin e for alzheimer &# 39 ; s disease . ann . pharmacotherapy 2005 , 39 ( 12 ): 2073 - 80 fay m j , bush m j , verlangieri a j : effect of aldonic acids on the uptake of ascorbic acid by 3t3 mouse fibroblasts and human t lymphoma cells . gen . pharmacol . 1994 , 25 ( 7 ): 1465 - 69 kaplan b , gonul b , dincer s , dincer kaya f n , babul a : relationships between tensile strength , ascorbic acid , hydroxyproline , and zinc levels of rabbit full - thickness incision wound healing . surg . today 2004 , 34 ( 9 ): 747 - 51 kurilich a c , jefferey e h , juvik j a , wallig m a , and klein b p : antioxidant capacity of different broccoli ( brassica oleracea ) genotypes using the oxygen radical absorbance capacity ( orac ) assay . j . agric . food chem . 2002 , 50 ( 18 ): 5053 - 57 landmark k : could intake of vitamins c and e inhibit development of alzheimer dementia ? tidsskr nor laegeforen 2006 , 15 ( 8 ): 159 - 61 lehr h a , frei b , arfors k e : vitamin c prevents cigarette smoke - induced leukocyte aggregation and adhesion to endothelium in vivo . pnas 1994 , 91 ( 16 ): 7688 - 92 marionnet c , vioux - chagnoleau c , pierrard c , sok j , asselineau d , bernerd f : morphogenesis of dermal - epidermal junction in a model of reconstructed skin : beneficial effects of vitamin c . exp . dermatol . 2006 , 15 ( 8 ): 625 - 33 moore j , hao z , zhou k luther m , costa j , y l : carotenoid , tocopherol , phenolic acid , and antioxidant properties of maryland - grown soft wheat . j . agric . food chem . 2005 , 53 ( 17 ): 6649 - 57 prior r l , cao g : antioxidant capacity and polyphenolic components of teas : implications for altering in vivo antioxidant status . proc . soc . exp . biol . med . 1999 , 220 ( 4 ): 255 - 61 schauss a g , xianli w ., prior r l , ou b , huang d , owens j , agarwal a , jensen g s , hart a n , shanbrom e : antioxidant capacity and other bioactivities of the freeze - dried amazonian palm berry , euterpe oleraceae mart . 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