Patent Application: US-39352009-A

Abstract:
an improved wound dressing using a honey composition is provided . preferably , the honey composition includes buckwheat honey .

Description:
honeys of different floral sources were tested for their ability to reduce oxidant levels as described above , in order to select the most active variety in this respect for use in wound - management products to be developed . testing included two varieties , which had already been applied in commercial wound - healing products , i . e . 1 ) unifloral manuka honey , highly valued for its medicinal properties and subject of several studies by the scientific community 1 , 7 , 10 , and 2 ) special multifloral honey from chile , selected by sociedad apicola verkruisen for application to wounds on basis of high glucose oxidase content ( as will be explained in the discussion section ). other varieties tested concerned two samples of american buckwheat honey very dark - brown in color , and two exotic varieties coming from hawaii , i . e . brown macadamia honey , and almost white rare kiawe honey . finally , a canadian mixture of clover and alfalfa honey as another representative of light - colored types besides hawaiian kiawe honey completed the series . a mixture of clover ( trifolium species ) and alfalfa ( medicago saliva ) honey came from canada ( golden acres co ,, three hills , alberta ), and was coded ca . two samples of american buckwheat honey ( fagopyrum esculentum ), aa and ab , supplied by bee keepers in the states of new york and north dakota , respectively , were obtained from dutch gold honey inc . ( lancaster , pa ., u . s . a .). chilean honey ( ch ) from the maule region , miel de abeja organica , was received from sociedad apicola verkruisen ( san javier , chile ). active manuka ( leptospermum scoparium ) honey ( m ), supplied by comvita ( bay of plenty ) was purchased in new zealand . from hawaii two samples were obtained , i . e . ha , macadamia honey ( macadamia integrifolia ; hawaii island honey co . ), and hb , kiawe honey ( prosopis pallida ) supplied by volcano island co . different varieties of honey were tested in bioassays , which are commonly used for anti - inflammatory activity screening . the following bioassays were performed , which have been described in detail , previously . 30 honey investigated as inhibitor of ros produced by pmns using a luminol - dependent chemiluminescence assay this chemiluminescence assay concerns physiologically relevant in vitro assessment of potential anti - inflammatory effects on basis of inhibition of ros production by inflammatory cells . honey samples were tested for their ability to inhibit production of reactive oxygen species ( ros ) by zymosan - activated human neutrophils ( pmns ) using luminol as chemiluminescent probe . reaction of luminol with ros , in particular hypochlorite anion ( ocl 31 ), results in formation of an excited oxidation product , which emits light ( chemiluminescence ) upon returning to its ground state . briefly , in a 96 - wells plate , freshly isolated human pmns 31 were stimulated by addition of opsonized zymosan . the resulting production of ros was detected as luminol - dependent chemiluminescence using a titertek luminoskan luminometer . chemiluminescence was monitored for 0 . 5 seconds every two minutes over a 30 - minute period at 37 ° c . peak levels of chemiluminesence measured for honey samples and controls ( identical incubations without honey present ) were used to calculate inhibition of ros production . 32 although not causing tissue damage as such , superoxide anion ( o 2 •− ) may eventually lead to detrimental effects by conversion of o 2 •− into hydroxyl radical , hypochlorite anion , and / or peroxynitrite . thus , scavenging of superoxide anion may contribute to honey &# 39 ; s wound - healing properties . different from the assay mentioned above , which is primarily focused on inhibition of ros production , thus pmn functioning , the scavenging assay concerns a situation with superoxide anion already present . the capacity of honey samples to scavenge superoxide anion was determined in a cell - free system as inhibition of chemiluminesence ; lucigenin was used as chemiluminescent marker , specifically detecting superoxide anion . briefly , in a 96 - wells plate , production of superoxide anion was initiated by addition of xanthine oxidase to a mixture of hypoxanthine , lucigenin and honey sample . the chemilumineseence signal generated was monitored for 0 . 5 seconds every three minutes during 30 minutes , using a fluoroskan ascent fl luminometer . ultimately , activity of the honey samples was calculated from the part of the chemilumineseence signal that was inhibited by superoxide dismutase ( sod ). 30 testing honey for inhibition of human complement using a hemolytic assay activity of different honey varieties concerned inhibition of human complement activated via the classical pathway , and was determined using a microtiter plate - method . 33 briefly , serial dilutions of honey samples were prepared in veronal buffer supplemented with calcium ( ca 2 + ) and magnesium ions ( mg 2 + ), and incubated with human pooled serum ( hps ) as source of complement for 30 minutes . subsequently , sheep erythrocytes ( she ) were added , which had been sensitized by incubation with amboceptor , as classical pathway complement activity can only be triggered by particles or cells coated with immunoglobulins . activation of complement eventually results in formation of mac ( membrane attach complex ) causing lysis of she . the amount of hemoglobin released , spectrophotometrically determined at 405 nm using an automatic elisa reader , served as measure for classical pathway complement activity . in general , inhibitory activity is expressed as ic50 , which is the amount of sample per ml ( mg / ml ) in the test system ( bioassay ) giving 50 % inhibition , thus low ic50 values representing strong activities . however , diagrams showing test results as ic50 often cause confusion by low bars indicating high activity and high bars representing minor effects . therefore , activities have been presented here as ric50 ( reciprocal ic50 ), 1 / ic50 in ml / g , which is the volume ( ml ) to be added per gram of sample giving 50 % inhibition . in this way , samples with stronger activities requiring more dilution ( increased volume ) show higher ric50values . high bars now corresponding with strong activities greatly improves interpretation of test results as presented in bar diagrams . ric50 values presented are the mean ± standard deviation ( sd ). to prevent growth of high osmolarity - resistant yeast species , the water content of honey should not be more than 20 %. although eventual honey products may be gamma - ray sterilized , growth of yeasts prior to sterilization may affect honey &# 39 ; s medicinal properties . the amount of water present was determined according to the european pharmacopoeia 34 by measurement of refractive index ( ri ), which increases with increasing sugar concentration . moisture content was derived from ri corrected for temperature (+ 0 . 00023 /° c . ), using a table of ri values ( at 20 . 0 ° c .) with corresponding water content listed . since acidity may be a factor contributing to wound healing , ph and free acid content were also determined . a solution of 25 gram of buckwheat honey in 75 ml of carbon dioxide - free demineralized water was used for determination of ph . the latter was measured as the potential difference ( voltage ) between a glass electrode and silver chloride ( agcl ) reference electrode as described in the european pharmacopoeia . 34 free acids were quantified by potentiometric acid - base titration with potassium hydroxide solution of known concentration as described in harmonized methods of the international honey commission . 35 in general , the end - point is determined using a visual indicator such as phenolphthalein , colorless in acid solution , but turning into pink above ph 8 . 2 . however , for buckwheat honey with its dark - brown color , a visual indicator could not be used . therefore , the point of equivalence was determined at ph 8 . 3 by potentiometric measurement ( see above ). free acid content of honey is expressed in meq / kg ( milliequivalents / kg ), which is the volume ( in ml ) of 1 m potassium hydroxide solution to be added to 1000 g of honey for neutralization of acids present . thin layer chromatography is a rapid analytical method for detection of separate compounds present in complex mixtures . after application of a multicomponent sample to a tlc plate at the start position , components of interest are separated by a solvent ( mobile phase ) migrating through the thin sorbent layer ( stationary phase ). the retention factor or r r is defined as the traveling distance by a compound from the start ( fig1 : a ) divided by the distance allowed for solvent migration ( from start to solvent front ; fig1 : b ). constituents are detected as colored spots under ultraviolet light and / or after spraying the chromatogram with specific reagents ( fig1 ). thus , tlc enables characterization of compounds by r f value and specific color reactions . consisting of mainly sugars , honey as such cannot be subjected to tlc analysis of phenolic components , directly . the latter constituents present in minor amounts can only be detected after concentration and without sugars interfering . therefore , phenolic constituents were extracted from honey prior to tlc analysis , as follows : honey ( 25 g ) was dissolved in 75 ml of water , and the solution was extracted with ethyl acetate ( 3 × 50ml ). the combined ethyl acetate extracts were dried over anhydrous magnesium sulfate . after filtration , and evaporation of the solvent under reduced pressure , the residue was taken up in 10 ml of ethanol ( 96 %) and subjected to tlc analysis . ethanol solutions containing phenolic constituents ( see above ) extracted from american ny buckwheat honey ( aa ) and chilean honey ( ch ) were applied in 30 μl amounts to tlc plates pre - coated with silica gel . the solvent system used was acetonitrile / water ( 87 : 13 ; saturated chamber ). after development of the chromatogram , the plate was sprayed with npr ( natural product reagent ; 2 - aminoethyldiphenyl borinate ), and examined under ultraviolet light of 366 nm . npr specifically detects phenolic constituents , including flavonoids , which become visible as blue and green fluorescent spots . spraying ( another ) chromatogram with dpph ( 2 , 2 - diphenyl - 1 - picrylhydrazyl radical ; 0 . 2 mg / ml dissolved in absolute ethanol ) showed the presence of constituents with antioxidant activity as yellow spots on a purple background ( examination in daylight ). dpph is a relatively stable purple - colored radical that turns into yellow dpphh by acceptance of hydrogen atom donated by antioxidants . different honey varieties were tested in vitro for activities leading to reduction of oxidant levels , i . e . inhibition of ros production , scavenging of superoxide anion , and inhibition of human complement . all honey samples tested showed inhibition in the bioassay for ros produced by activated human pmns with activities ranging from 160 and 130 ml / g ( buckwheat honey from new york , and north dakota , respectively ) to 40 ml / g ( hawaiian kiawe honey ) as shown in fig2 . no differences were found between chilean and manuka honey showing a ric50 value around 110 ml / g . differences in superoxide anion scavenging capacity were found to be much more pronounced ( fig3 ). again , highest activity by far was shown by ny buckwheat honey ( ric50 : 290 ml / g ), exceeding the ric50 value of least active hawaiian kiawe honey ( 10 ml / g ) by factor almost 30 . although showing half the activity as determined for the new york sample , in comparison with the other samples investigated superoxide anion scavenging capacity of north dakota buckwheat honey ( ric50 : 150 ml / g ) can still be considered strong . no significant difference was found between activities determined for chilean ( 59 ml / g ) and manuka honey ( 48 ml / g ). results of testing honey samples for inhibition of human classical pathway ( cp ) complement activity are presented in fig4 . although the strongest inhibition was shown by ny buckwheat honey ( ric50 : 120 ml / g ), this activity was not significantly different from nd buckwheat ( 102 ml / g ), hawaiian macadamia ( 115 ml / g ) and manuka honey ( 111 ml / g ). in this assay , the chilean sample ( ric : 33 ml / g ) was found to score worse than manuka , and equal to hawaiian kiawe honey ( ric50 : 34 ml / ml ). american buckwheat honey from new york showed the most pronounced activities ( fig2 - 4 ), that is the inhibition of human complement , which results in reduced formation of factors that attract and stimulate pmns , as well as scavenging of oxidants produced by pmns upon activation . the in vitro results obtained were encouraging and honey may exert these activities in vivo , as well . however , this was not assessed in this study . on basis of superior in vitro activities , and after it had been established that heavy metals , pesticides and antibiotics were all below detection limits or present in acceptable amounts , ny buckwheat honey was selected to be used in wound - healing products , i . e . melmax ® and meldra ® ( dermagenics europe bv , kaatsheuvel , the netherlands ), and subjected to further research . in addition to testing for biological activities resulting in decreased levels of radicals and oxidants , and analysis of contaminants for safety purposes , some other parameters were determined that may also contribute to wound - healing effects of ny buckwheat honey , and quality of eventual products . refractive index ( ri ) of ny buckwheat honey measured at 22 . 1 ° c . was n d 1 . 4939 . correction for temperature resulted in n d 1 . 4944 , corresponding with a moisture content of 16 . 8 % ( w / w ), which is perfectly below the upper limit of 20 % as required by european legislation 36 , and european pharmacopoeia 34 . ny buckwheat honey was found to have relatively strong acid properties , represented by low ph and high free acid content . measurement of ph using a solution of ny buckwheat honey in water ( 25 % w / v ) showed ph 3 . 3 , which is considered low in comparison with the average ph 3 . 9 for non - tropical honeys with a typical range of 3 . 4 to 6 . 1 . 37 the ph represents - 10 log [ h + ], [ h + ] being the concentration of protons ( h + ions ) in moles per liter . free acid content is the amount of 1 m potassium hydroxide solution ( in ml ) required for neutralization of acid components present in 1 kg of honey . in contrast to strong mineral acids ( e . g . sulfuric acid ), acids in honey concern weak organic acids only capable of limited dissociation into protons , which results in lower | h + | thus higher ph values . neutralization by potassium hydroxide results in complete dissociation , however . so , free acid content concerns components being capable to split off protons , whereas ph is a measure for protons being actually present . in other words , the free acid content of honey is higher than can be derived from its ph measured . determination of free acids in ny buckwheat honey showed a content of 50 meq / kg , which is exactly the maximum allowed by european legislation towards honey used tor consumption . 36 showed blue and green - fluorescent spots , indicating phenolic compounds , including flavonoids , extracted from ny buckwheat honey ( fig1 : tlc - a1 ). detection of antioxidants by dpph showed the presence of some strong antioxidant constituents that immediately turned purple dpph into yellow ( fig1 : tlc - b1 ). in fig1 , a1 and a2 , and b1 and b2 refer to pans of two separate chromatograms a and b , which were developed under similar experimental conditions . by this , identical constituents of ny buckwheat honey on tlc - a1 and tlc - b1 , will have the same rf value as indicated by 1 - 4 ( fig1 ). since fluorescent constituents were found in this respect to correspond with those detected by dpph , phenolic compounds present in ny buckwheat honey were concluded to be major antioxidant principles , and such may account as well for antibacterial activity . 38 in addition , it was found that amounts of antioxidants in ny buckwheat honey ( fig1 : tlc - b1 ) greatly outmeasured those present in chilean honey ( fig1 : tlc - b2 ). the component indicated by 3 with r f 0 . 53 was found to be major antioxidant constituent of both ny buckwheat and chilean honey , although the latter contained a relatively minor amount , only slightly reducing dpph as observed by tlc ( fig1 : tlc - b2 ). the chilean honey investigated has also been used in wound care products , e . g . honeysoft ( mediprof b v , rijswijk , the netherlands ), but was selected on basis of high glucose oxidase content . ny buckwheat honey selected on basis of pronounced in vitro activities since elevated levels of reactive oxygen species may cause cell damage and inhibition of wound healing 24 , 25 , honeys of different floral sources were tested in vitro for activities , which lower levels of oxidants , radicals included . on this basis ny buckwheat honey showing superior effects was selected for medicinal application . in comparison with manuka and chilean honey , which have both been used in commercial wound - healing products , ny buckwheat honey was found to be a most strong scavenger of superoxide anion , in particular ( fig3 ), and most effective inhibitor of ros produced by stimulated human pmns ( fig2 ), ny buckwheat honey was also shown to inhibit classical pathway complement activity ( fig4 ), which results in limited formation of complement factors attracting and activating pmns . in the latter assay , however , pronounced inhibition was not restricted to ny buckwheat honey , only , as nd buckwheat . hawaiian macadamia , and manuka honey showed similar activities with differences not being significant . strict requirements regarding heavy metals , pesticides and antibiotics present have neither been included in the honey monograph of the european pharmacopoeia 34 , nor formulated for honey as food by european legislation 36 . nevertheless , to guarantee safety for wound - healing products eventually produced , ny buckwheat honey was tested for these contaminants , which were below detection limits or present in the amounts allowed for other food products . concerning quality , another parameter determined was moisture content . with 16 . 8 % ( w / w ) water present , ny buckwheat honey amply met the legislatory requirements . as consequence of its high osmotic value ( high sugar content ) in general , honey draws wound exudate to the wound surface , thus creating a moist environment , which results in a non - adherent interface between dressing and wound bed . 7 reports on the significance of high osmolarity for antibacterial activity of honey are controversial ( see below ). however , moisture content was not determined as antibacterial factor of ny buckwheat honey to be applied , but as control to exclude high - osmolarity resistant yeasts having affected its properties , prior to being processed into wound - healing products . the value of honey as an antibacterial has since long been recognized . hydrogen peroxide — already present ( residual ) and / or generated by glucose oxidase activity upon dilution of honey — as well as phenolic constituents are considered major antibacterial factors . 1 , 38 neither glucose oxidase , nor residual hydrogen peroxide could be detected in ny buckwheat honey selected for application to wounds . with sugar contents above 80 % ( w / w ), the high osmolarity of undiluted honey is sufficient to stop all microbial growth . 1 in practice , however , this may be of minor importance . dependent of the type of wound , dilution by exudate may eventually result in loss of osmotic antibacterial activity . 1 , 7 somewhat contradictory , high osmolarity has been claimed responsible for honey &# 39 ; s antimicrobial activity , if accompanied by low ph . 39 , 40 however , not ph , but free acid content of non - peroxide honey has been found to significantly correlate with antibacterial activity against staphylococcus aureus and micrococcus luteus . 41 in comparison with the average ph 3 . 9 reported for non - tropical honeys with ph ranging from 3 . 4 to 6 . 1 37 , ph 3 . 3 as measured for ny buckwheat honey can be considered low . furthermore , its free acid content was determined to be the maximum allowed by european legislation 36 , being 50 meq / kg . referring to above , the low value for ph , but even more the high free acid content 41 may be characteristics of ny buckwheat honey , contributing to antibacterial activity . lysed concentrates of platelets , pre - incubated at close to ph 5 . 0 ( as opposed to ph 7 . 0 ), have been found to contain increased levels of platelet - derived growth factor ( pdgf ), and showed increased capacity to stimulate fibroblast proliferation in vitro . 42 although such has not been studied , showing relatively strong acidic properties , ny buckwheat honey may promote healing of chronic wounds by effecting a low ph at the wound site , resulting in fibroblast proliferation . compared to other types of honey , buckwheat honey is a rich source of phenolic antioxidants . 43 since phenolic compounds also have antibacterial activity , 38 lack of glucose oxidase activity or hydrogen peroxide in ny buckwheat honey may well be compensated by phenolic constituents being present in relatively large amounts . after selection based on in vitro biological activity , and additional research regarding quality aspects , as described above , ny buckwheat honey was used in wound - healing products , which were tested in pilot clinical experiments . in a study including 21 burn patients with difficult to treat wounds , application of melmax ® ( a wound dressing impregnated with ny buckwheat honey and an ointment containing a synthetic blend of metal ions and citric acid 30 ) resulted in wound closure and control of microbial contamination . 44 in another study including 60 patients , it was shown that treatment of venous leg ulcers with melmax ® showed similar results as silver - based dressings , or even better . 45 as compared to honeys from some other floral sources , ny buckwheat honey showed pronounced in vitro activities leading to decreased levels of oxidants , radicals included . antioxidant principles in ny buckwheat honey are constituted by phenolic constituents , in particular . the relatively low ph and high free acid content determined for ny buckwheat honey are other characteristic features , which may also contribute to healing of chronic wounds . initial clinical pilot experiments have shown that melmax ® containing ny buckwheat honey was successfully applied to burn wounds and venous leg ulcers . although often mentioned as major wound - healing factors , residual hydrogen peroxide , or glucose oxidase could not be detected . instead , buckwheat honey was found to contain large amounts of antioxidant phenolic constituents that may also have antibacterial activity , thus compensating absence of hydrogen peroxide or its generation by glucose oxidase . in wound management , bacteria resistant to antibiotics are becoming an increasing problem . although results obtained so far are most promising , and indicate ny buckwheat honey to be an effective antimicrobial wound - healing product , final proof will only be provided by full clinical trials . 1 molan p c . re - introducing honey in the management of wounds and ulcers — theory and practice . ostomy / wound management 2002 ; 48 : 28 - 40 . 2 molan p c . the antibacterial activity of honey . 2 . variation in the potency of the antibacterial activity . bee world 1992 ; 73 : 59 - 76 . 3 gunther r t . the greek herbal of dioscorides . new york , ny : hafner ; 1934 ( reprinted 1959 ). 4 namias n . honey in the management of infections , surg infect 2003 ; 4 : 219 - 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