Patent Application: US-81248808-A

Abstract:
a method of modulating inflammation in an organism , which includes administering to an organism a composition including a therapeutic amount of an extract from the plant biota orientatis . several key components of the extract of biota orientalis have been identified that have also been shown to have an effect in dramatically reducing inflammatory responses .

Description:
to facilitate an understanding of the invention various terms and abbreviations are used and defined below : “ seq ” means a blend of new zealand green lipped mussel , abalone , shark cartilage powder and biota oil . “ bo ” means “ biota oil ” being an extract of the seeds of the plant biota orientalis . biota is an herb native to western china and north . korea and is known by a number of other names , such as thuja oriental &# 39 ; s , platycladus stricta , and platycladus oriental &# 39 ; s . simulated digests of shark cartilage , nzglm and abalone have been previously reported to have anti - inflammatory effects in a cartilage explant model of arthritis by reducing pge2 , gag and / or nitric oxide ( pearson et al ., 2007 ). the following data reports alterations in gene expression associated with conditioning cartilage explants with simulated digests of the combination of all four constituents ( seq ; seq sim ), and , to characterize their effects on il - 1 - induced pge 2 , gag , no , cell viability , and genetic expression of cox 1 , cox 2 , inos and aggrecan . front legs of market weight pigs ( 5 - 7 months old , 200 - 250 lbs ) were obtained from a local abattoir . legs were chilled on crushed ice until dissection . using aseptic technique , the intercarpal joint was opened and the cartilage surfaces exposed . a 4 mm dermal biopsy punch was used to take explants (˜ 0 . 5 mm thickness ; 11 - 15 mg / explant ) of healthy cartilage from the weight - bearing region of both articulating surfaces of the intercarpal joint . cartilage pieces were washed 3 times in dmem supplemented with nahco 3 . two cartilage discs were placed into each well of 24 - well tissue culture plates containing dmem supplemented with amino acids , sodium selenite , manganese sulfate , nahco 3 and ascorbic acid ( tcm - tissue culture medium ). plates were incubated at 37 ° c ., 7 % co 2 in a humidified atmosphere for up to 144 h . every 24 h media was completely aspirated into 1 ml microcentrifuge tubes and immediately replaced with control , conditioned and / or stimulated media ( described below ) before being returned to the incubator . the collected media was stored at − 80 ° c . until analysis . cartilage was harvested at the end of each experiment with one explant per well stained for cytotoxicity and the remaining cartilage immediately frozen at − 80 ° c . a simulated digestion procedure was developed to mimic the gastrointestinal processing of ingested dietary supplements . this type of approach has previously been used to improve the bio - assessment of putative nutraceuticals ( rininger et al ., 2000 ; pearson et al ., 2007 ). simulated digests were prepared using seq ( 0 . 85 g ), bo [ 2 . 5 ml ( 0 . 85 g )] and indo ( 0 . 074 g — a positive anti - inflammatory control ). each test substance was individually suspended in 35 ml of simulated gastric fluid ( 37 mm nacl , 0 . 03n ncl , 3 . 2 mg / ml pepsin ), and shaken at 37 ° c . for 2 h ( rininger et al ., 2000 ). after this , solution acidity was neutralized by adding an equinormal volume of 2 . 2 n naoh ( 1 . 15 ml ). to this was added 36 . 15 ml of simulated intestinal fluid ( rininger et al ., 2000 - 30 mm k 2 hpo 4 , 160 mm nah 2 po 4 ; 20 mg / ml pancreatin ; ph adjusted to 7 . 4 ) and the resultant mixture shaken in a 37 ° c . incubator for a further 2 h . a “ blank ” was prepared using identical methodology but without including any test substance . appropriate volumes of gastric and intestinal fluid were derived from those approximated in a human stomache ( marciani et al ., 2005 ). upon completion of the 4 - hour incubation , simulated digests of seq ( seq sim ) bo ( bo sim ) and indomethacin ( indo sim ) were centrifuged at 3 , 000 × g for 25 min at 4 ° c . the supernatant was decanted and centrifuged a second time at 3 , 000 × g for 15 min at 4 ° c . the resulting supernatant was warmed to room temperature and filtered ( 0 . 22 μm ) to remove particulates . this filtrate was further fractioned with an ultrafiltration centrifuge unit with a 50 kda molecular weight cut - off , ( amiconultra , millipore , mississauga on ), spinning at 3 , 000 × g for 25 min ( room temperature ). filtered simulated digest was stored at 4 ° c . until use for a maximum of 7 days . seq sim was prepared as explained above . explants from 12 pigs were prepared as previously described , and maintained in unconditioned media for the initial 24 h . at 24 hours post - culture , seq sim , bo sim ( 0 , 0 . 06 or 0 . 18 mg / ml ) or indo sim ( 0 . 02 mg / ml ) was added to tcm ( conditioned media ). conditioned media was refreshed every 24 hours for the duration of the experiment . at 72 hours post - culture , and every 24 hours thereafter , explants were stimulated with il - 1 ( 0 or 10 ng / ml ; medicorp , montreal , quebec ; cat . # phc0813 ). explants from each animal were exposed to each treatment in duplicate . explants were cultured for a total of 120 h . media was analyzed for [ pge 2 ], [ gag ], [ no ]. one explant per treatment was collected into sterile phosphate , buffered saline ( pbs ) and immediately stained for cell viability ( see below ). the second explant was frozen at − 80 ° c . for rna extraction ( see below ). pge 2 concentration of tcm was determined using a commercially available pge 2 elisa , kit ( the kit has 7 % cross - reactivity with pge 1 ) ( amershan , baie d &# 39 ; urfé , québec ). plates were read using a victor 3 microplate reader ( perkin elmer , woodbridge on ) with absorbance set at 405 nm . pge 2 standard curves were developed for each plate , and a best - fit 3 rd order polynomial equation with r 2 ≧ 99 was used to calculate pge 2 concentrations for standards and samples from each plate . no concentration of tissue culture media was determined by the griess reaction ( shen et al ., 2005 ). plates were read using a victor 3 microplate reader with absorbance set at 530 nm . sodium nitrite standard curves were developed for each plate , and a best - fit linear regression equation with r 2 ≧ 99 was used to calculate no concentrations , which were compared with the nitrite standard . total rna was extracted from cartilage explants using a modified trizol procedure ( chan et al ., 2005 ). frozen cartilage from each animal was pooled according to conditioning and stimulation , and homogenized in tri - reagent ( 100 mg tissue / ml ; sigma , mississauga on ). chloroform was added to extract rna followed by vigorous agitation and 2 - min incubation at room temperature . sample was then centrifuged ( 12 , 000 × g , 15 min ) and rna was precipitated with an equal volume of 70 % ethanol ( depc ). rna precipitate was applied to an rneasy mini column ( qiagen , valencia calif ., usa ) and rna was purified according to manufacturer instructions . for each pooled sample , 1 μg total rna was converted to single stranded cdna using moloney murine leukemia virus ( mmlv ) reverse transcriptase ( invitrogen , burlington on ) according to manufacturer instructions . single - strand cdna was quantified by uv spectrophotometry and diluted with depc - h 2 o to a final concentration of 10 ng / μl . primers for porcine inos ( granja et al ., 2006 ), cox1 / 2 ( blitek et al ., 2006 ), aggrecan ( fehrenbacher et al ., 2003 ) and β - actin ( housekeeping gene ; nishimoto et al ., 2005 ) ( table 1 ) were prepared ( laboratory services division , university of guelph ) and stored at − 20 ° c . until use . cartilage samples from seq sim and bo sim were evaluated for changes in gene expression , together with cartilage cultured under identical conditions previously with the other 3 components of seq ( see pearson et al ., 2007 for detailed culture , conditions ). twenty five microliter pcr reactions were performed in triplicate using an abi prism 7000 sequence detection system ( perkin - elmer ). amplification of 50 ng of each cdna sample was detected using sybr - rox ( invitrogen , burlington on ) and compared to a standard curve of pooled cdna containing equal amounts of cdna from each sample . a 1 . 5 % agarose electrophoresis gel was used to confirm pcr products . expression of each gene of interest ( g ) in each sample was compared to amplification of β - actin ( β ), and calibrated to unstimulated control explants ( ie . fold change for calibrator = 1 ). fold change in expression ( δg / δβ ) is presented in arbitrary units . cell viability was determined using a commercially available viability staining kit ( invitrogen ; burlington on ) ( pearson et al ., 2007 ). briefly , explants were washed in 500 ul pbs and placed into a 96 - well microtitre plate ( one explant per well ), and were incubated in 200 ul of stock stain ( 4 μm c - am ; 8 μm ethd - 1 ) for one hour at room temperature . the plate was read from the bottom of each well using 10 horizontal steps , 3 vertical steps , and a 0 . 1 mm displacement . c - am and ethd - 1 fluorescence in live and killed explants were obtained with excitation / emission filters of 485 / 530 nm and 530 / 685 nm , respectively . data from analysis of tissue culture media and viability are presented as means ± standard error . means of replicates from each treatment / animal were analyzed using two - way repeated measures analysis of variance comparing each treatment with unconditioned controls and indomethacin - conditioned controls . viability data were analyzed using the student &# 39 ; s t - test , individually comparing stimulated controls with all other treatments . when a significant f - ratio was obtained , the holm - sidak post - hoc test was used to identify significant differences between treatment and / or time . significance was accepted if p ≦ 0 . 05 . due to low cellularity of cartilage explants , it was necessary to pool rna from explants exposed to the same conditioning and stimulation in order to extract sufficient rna for a reverse transcription reaction . thus , pcr data are presented in the text as a mean change in gene expression ( calibrated to controls ) relative to β - actin ± coefficient of variation for the assay . a calibrated fold expression change ≧ 2 is considered to be biologically relevant ( yang et al ., 2002 ; schena et al ., 1995 ) and are discussed in the text as significant differences . cox 1 ( fig1 , a and b ): 1 l - 1 stimulation of control explants resulted in a 35 % increase in cox 1 expression compared with unstimulated controls . cox 1 expression was decreased by exposure to indo sim by 98 and 91 . 5 % in unstimulated and stimulated explants , respectively . all constituents of seq reduced cox 1 expression in unstimulated explants ( range : 76 - 95 % inhibition ). importantly , it was observed that bo sim ( 0 . 06 mg / ml ) was the most effective cox 1 inhibitor , reducing cox 1 expression by 95 % in both unstimulated and stimulated explants . in addition , it was observed that seq sim ( 0 . 06 and 0 . 18 mg / ml ) reduced cox 1 expression in unstimulated explants by 90 and 80 %, respectively . in il - 1 stimulated explants , seq sim ( 0 . 06 and 0 . 18 mg / ml ) inhibited cox 1 expression by 57 and 76 %, respectively . the least effective cox 1 inhibitor in il - 1 - stimulated explants was nzglm ( 0 . 18 mg / ml ), which increased cox 1 expression by 62 %. fold change in cox 1 for all samples was & gt ; 2 and therefore not considered significant . cox 2 ( fig2 , a and b ): stimulation of control explants resulted in a significant 4 . 3 - fold increase in cox 2 expression . indo sim reduced expression of cox 2 by 44 and 47 % in unstimulated and stimulated explants , respectively . fold increase in cox 2 for indo sim - conditioned , il - 1 - stimulated explants was significant ( 2 . 3 ). abalone ( 0 . 18 mg / ml ) significantly increased cox 2 expression in unstimulated explants , showing similar effect on cox 2 ( 3 . 7 - fold ) as il - 1 . all other constituents decreased cox 2 expression in unstimulated explants ( range : 56 - 90 %). il - 1 - stimulation resulted in a significant increase in cox 2 expression in those explants conditioned with indo sim ( 2 . 3 - fold ), seq sim ( 0 . 06 mg / ml ; 2 . 0 - fold ), nzglm sim ( 0 . 18 mg / ml ; 28 . 2 - fold ), and ab sim ( 0 . 18 mg / ml ; 41 . 5 - fold ). all other constituents prevented a significant increase in il - 1 - induced cox 2 expression ; the most effective inhibitor was bo sim ( 0 . 06 mg / ml ) which inhibited cox 2 expression by 92 %. inos ( fig3 , a and b ): stimulation of control explants by il - 1 resulted in a 287 - fold increase in inos expression . indo sim conditioning had no effect on inos in unstimulated explants . in il - 1 - stimulated explants , indo sim conditioning augmented the effect of il - 1 on inos expression ( 725 - fold increase ). seq and all of its individual constituents significantly increased inos expression in unstimulated explants ( range : 39 - 2486 - fold increase ). il - 1 - stimulation resulted in a significant increase in inos expression in all conditioned explants . however , compared with il - 1 - stimulated controls , inos was significantly inhibited by both doses of seo sim in a dose - dependent manner ( 60 and 89 % inhibition for 0 . 06 and 0 . 18 mg / ml , respectively ). bo sim ( 0 . 06 mg / ml ) and ab sim ( 0 . 18 mg / ml ) also significantly inhibited il - 1 - induced inos expression by 55 and 12 %, respectively . aggrecan ( fig4 , a and b ): stimulation of control explants with il - 1 resulted in a slight , non - significant decline in aggrecan expression . conditioning of unstimulated explants with indo sim resulted in 58 - fold increase in aggrecan . this increase was completely abolished by stimulation of indosim - conditioned explants with il - 1 . seq and all of its constituents significantly increase aggrecan expression in unstimulated explants . seq sim increased aggrecan expression in unstimulated explants in a dose - dependent manner ( 42 . 8 and 215 . 7 - fold increase for 0 . 06 and 0 . 18 mg / ml , respectively ). stimulation of conditioned explants with . il - 1 rebutted in significant increase in aggrecan expression in seq and all of its constituents , with the exception of sc sim ( 0 . 18 mg / ml ; 1 . 4 - fold increase ). pge 2 ( fig5 , a and b ): stimulation of control explants with il - 1 ( 10 ng / ml ) resulted in a significant increase in media [ pge 2 ] over the 48 h stimulation period , resulting in a significant difference between stimulated and unstimulated controls ( p = 0 . 03 ). indo sim ( 0 . 02 mg / ml ) significantly reduced media [ pge 2 ] in il - 1 stimulated and unstimulated explants compared with stimulated and ustimulated controls , respectively . there was no il - 1 - induced increase in media [ pge 2 ] in explants conditioned with indo sim . stimulation with il - 1 of explants conditioned with seq sim ( 0 . 06 and 0 . 18 mg / ml ) did not increase media [ pge 2 ]. media [ pge 2 ] was significantly lower in these explants compared with stimulated and unstimulated control explants ( fig5 , a ). in unstimulated explants media [ pge 2 ] was significantly lower in explants conditioned with seq sim ( 0 . 06 and 0 . 18 mg / ml ) than in unstimulated controls ( fig5 , b ). there was no significant difference in media [ pge 2 ] between seq sim ( 0 . 06 and 0 . 18 mg / ml ) and indo sim in both il - 1 - stimulated and unstimulated explants . there was no increase in media ipge 2 ] subsequent to il - 1 exposure in explants conditioned with bo sim ( 0 . 06 and 0 . 18 mg / ml ) ( fig5 , a ). conditioning of il - 1 - stimulated explants with bo sim ( 0 . 18 mg / ml ) resulted in a significantly lower media [ pge 2 ] than stimulated controls . there was no significant effect of bo sim on unstimulated explants ( fig5 , b ). no : there was no significant change in media [ no ] in unstimulated control explants . exposure of control explants to il - 1 ( 1 ong / ml ) resulted in a significant elevation of media [ no ] at 24 ( 1 . 21 ± 0 . 1 μg / ml ) and 48 h ( 1 . 06 ± 0 . 1 μg / ml ). there was no significant effect of indo sim on [ no ] in stimulated or unstimulated explants ( fig7 ). these experiments assist in describing effects of the simulated digest of seq on cox 1 , cox 2 , inos , and aggrecan gene expression . the gene expression data can then be used to make predictions about the mechanism of action of seq . alterations in gene expression observed in il - 1 - stimulated control explants showed a pattern consistent with an inflammatory response . il - 1 stimulation resulted in a small , non - significant increase in cox 1 expression coupled with a significant increase in cox 2 expression , as has been reported by other authors ( kydd et al ., 2007 ). as shown , indo sim showed a cox 1 : cox 2 inhibition profile of about 2 : 1 , which is consistent with its classification as a cox 1 / 2 inhibitor ( gerstenfeld et al ., 2003 ). we have also shown that indo sim does not inhibit il - 1 - induced inos expression , consistent with reports by other authors ( palmer et al ., 1993 ). nor did it influence il - 1 - mediated aggrecan expression in il - 1 - stimulated explants , an effect that has been reported in mechanically stressed cartilage explants ( limoto et al ., 2005 ). these data characterize indomethacin as an effective anti - inflammatory predominately through cox inhibition : its inability to reduce il - 1 - mediated aggrecan expression and its augmenting effect on il - 1 - mediated inos expression , however , suggest that cartilage exposed to indomethacin would continue to degenerate through decline in matrix formation and would suffer from increased nitric oxide - mediated cell death . indeed these adverse effects have been reported in arthritic dogs using prophylactic indomethacin ( hungin and kean 2001 ), and indomethacin is associated with worsening of some pathophysiological indicators of arthritis in humans ( rashad et al ., 1989 ; huakinsson et al ., 1995 ). when indo sim was applied to cartilage explants in the current study , there was an increase in il - 1 - mediated no production , but this was not coupled with a decrease in cell viability . the relative inhibitory profile of seq sim on cox 1 : cox 2 expression was approximately 1 : 1 at both doses . in the experiments described herein , seq sim at the lower dose was comparable to indo sim as a cox 2 inhibitor , whereas the higher dose was a more effective inhibitor of cox 2 than indo sim . it is therefore predicted that seq should effectively inhibit pge 2 production by il - 1 - stimulated explants . this inhibition was observed in the tissue culture explant experiment . inhibition of il - 1 - mediated pge 2 production by seq sim - conditioned cartilage explants was significant at both doses , and was not statistically different from pge 2 inhibition by indo sim . this provides an explanation for the observed clinical benefit of seq in relieving pain in arthritic patients ( rukwied et al ., 2007 ; zhao et al ., 2007 ). earlier publications have reported that sc sim and nzglm sim inhibit pge 2 production by il - 1 - stimulated cartilage explants ( pearson et al ., 2007 ), and the data in this application shows that bo sim also has this effect . however , it is of interest that , with the exception of sc sim ( 0 . 18 mg / ml ), cox 2 inhibition by the most effective dose of seq sim is stronger than any single constituents alone . this points to a synergistic relationship between the constituents . given the effective pge 2 - inhibiting , and related cox - inhibiting properties of seq sim , the effects of seq sim on inos were investigated . with a standard ‘ nsaid - like ’ mechanism it is predicted that seq would also augment inos expression in il - 1 - stimulated explants . in fact , the opposite was true , and seq sim was found to significantly and strongly inhibit inos expression . the effect of il - 1 on cellular expression of inos and cox 2 is differentially regulated through activation of at least 2 mitogen activated protein kinases ( mapks ) ( lapointe and isenovi 1999 ). net expression of inos and cox 2 are at least partially dependent on the relative amounts of pericellular no and pge 2 ( shin et . al ., 2007 ). thus , products which increase pericellular no can effectively downregulate expression of cox 2 , and vice versa ( shin et al ., 2007 ; kim et al ., 2005 ). this provides some explanation as to why seq sim showed a significant inhibitory effect on inos while many of the individual constituents , including shark cartilage , biota and nzglm sim ( 0 . 18 mg / ml ), actually upregulated expression of inos . seq is capable of effectively downregulating rna for inos and cox 2 . its effect on inos and cox 2 appears to be due to synergy between its four constituents , but it may be related to post - translational inhibition of no production ( pearson et al ., 2007 ). models of cartilage inflammation in horses are widely reported , and include intra - articular challenges such as lipopolysaccharide ( jacobsen et al ., 2006 ), freunds complete adjuvant ( toutain and caster 2004 ) or na - monoiodoacetate ( welch at al ., 1991 ); or surgical disruptions including creation of osteochondral fragments ( friable et al ., 2007 ), focal contusion impact injuries ( bolam et al ., 2006 ) and ligamentous transsection ( simmons et al .,. 1999 ). while these models capably demonstrate maximal activation of a complexity of inflammatory mechanisms within cartilage and associated subchondral bone and soft tissues , they represent a predominately traumatic inflammatory response . they are less representative of the more subtle biochemical , functional and pathophysiological changes in incipient or sub - acute articular inflammation that characterize most cases of lameness in racing horses ( steel et al ., 2006 ). while non - steroidal anti - inflammatory drugs ( nsaids ) and corticosteroids remain important therapeutic resources for treatment of overt clinical lameness , nutraceuticals are becoming widespread as a therapeutic and prophylactic management strategy , for horses with low - grade , sub - acute articular damage and for those at risk of developing articular problems ( trumble 2005 ; neil et al ., 2005 ). most research reported on the efficacy and / or safety of these products in arthritis uses in vitro models ( pearson et al ., 2007 ; chan et al ., 2006 ), or traumatic injury or clinical in vivo research in non - equine species ( mccarthy at al ., 2006 ; cho et al ., 2003 ). though useful as screening tools , in vitro models cannot account for the systemic effects of a dietary product which may influence outcomes in the articular space the objectives of this section are to a ) produce and characterize a reversible , sub - clinical model of il - 1 - induced intra - articular inflammation in the horse with respect to pge 2 and no production , and gag release from cartilage ; and b ) to apply this model to the evaluation of seq in mammals , particularly in horses . diets : seq powder was prepared by combining abalone ( ab ), new zealand green lipped mussel ( nzglm ), shark cartilage ( sc ) and biota oil ( interpath pty ltd , australia ) according to the composition provided in table 2 . seq mixed ration was prepared by combining seq powder ( 10 g / kg ), molasses ( 20 g / kg ) and flavoring ( essential sweet horse essence d 2344 . essentials inc . abbotsford , bc .) ( 1 g / kg ) to a sweet feed horse ration ( table 2 ), and blending in a diet mixer in 5 kg batches until fully mixed . control ration ( con ) was prepared using the same sweet feed diet blended with molasses (˜ 20 g / kg ) and flavoring ( 1 g / kg ). horses : 11 healthy horses without signs of articular inflammation ( 3 thoroughbred , 8 standardbred ; age 5 - 12 years ; 10 geldings , 1 mare ) were randomly allocated to either group a ( seq ; 1 . 5 kg / day ; n = 6 ) or group b ( con ; 1 . 5 kg / day ; n = 5 ). the 28 - day experiment consisted of two phases - phase 1 : pretreatment ( 14 days ); phase 2 : treatment ( 14 days ). supplementation began on day 0 and continued for the duration of the experiment ( fig6 ). sample collection occurred on days 0 ( pre ), 14 ( inj - 1 ), 15 ( 2 samples : inj - 2 — taken immediately before injection ; inj - 2 - 2 — taken 8 h post - injection ), 16 ( day 1 ), 18 ( day 3 ), 21 ( day 7 ) and 28 ( day 14 ); on these days blood was collected from the jugular vein , and synovial fluid was sampled from both intercarpal joints by aseptic arthrocentesis ( see below ). an inflammatory challenge — recombinant interleukin - 1β ( il - 1 )— was injected into the left or right intercarpal joint on day 14 ( inj - 1 ; 10 ng in 500 μl sterile saline ) and 15 ( inj - 2 ; 100 ng in 500 μl sterile saline ). an equal volume of sterile saline was injected into the contralateral intercarpal joint . joint circumference as an indicator of joint effusion was measured with a tape measure at each sampling of joint fluid . all horses were turned out in paddocks during the day and housed in box - stalls overnight . they were bedded on wood shavings and offered hay , water , and mineral salts ad libitum . all procedures were approved by the university of guelph animal care committee in accordance with guidelines of the canadian council on animal care . arthrocentesis : the knees of both the left and right legs were shaved , and the area aseptically prepared using chlorhexadine ( 4 %), and rinsed with 70 % isopropyl alcohol . a sterile 22 gauge , 1 . 5 ″ needle was inserted into the lateral aspect of the left intercarpal joint . a 3 cc sterile syringe was then attached , and approximately 1 . 5 - 2 ml of synovial fluid was aspired and immediately injected into a sterile k 2 - heparin vacutainer . the procedure was then repeated for the right intercarpal joint . on days 14 ( inj - 1 ) and 15 ( inj - 2 ), il - 1 ( 500 μl ) was injected into either the right or left intercarpal ( 500 μl saline injected into contralateral joint ) after aspiration of synovial fluid and before removal of the needle hub . approximately 1 . 5 ml of synovial fluid was removed from the vacutainer and placed into a microcentrifuge tube and spun at 11 , 000 × g for 10 minutes to remove cellular debris . supernatant was placed into another microcentrifuge tube containing 10 μg indomethacin , and frozen at − 80 ° c . until analyzed for pge 2 , gag and no . indomethacin was added to synovial fluid after it was collected in order to prevent further formation of pge2 during storage of samples . the remaining ˜ 0 . 5 ml synovial fluid was sent to the animal health laboratory ( university of guelph ) for cytological analysis . 1 . 0 - 1 . 5 ml of fluid was removed from the vacutainer for pge 2 , no and gag analysis ( see below ), and approximately 0 . 5 ml was analyzed for total nucleated cell count ( coulter z2 counter ; beckman coulter canada inc . mississauga on ), protein ( refractometer ) and cell differential ( on 100 nucleated cells ) at the animal health laboratory . synovial fluid was thawed to room temperature then incubated with 20 μl hyaluronidase ( 10 mg / ml ) on a tube rocker for 30 minutes at 37 ° c . to digest hyaluronic acid . sample was then diluted 1 : 2 with formic acid ( 0 . 1 %), and centrifuged 12 , 000 × g for 10 minutes . the supernatant was decanted and analyzed for pge 2 by a commercially available elisa kit ( ge amersham , baie d &# 39 ; urfé , québec ). pge 2 was extracted from the sample using provided lysis reagents to dissociate pge 2 from soluble membrane receptors and binding proteins , and then quantified according to kit protocol . plates were read using a victor 3 microplate reader ( perkin elmer , woodbridge on ) with absorbance set at 450 nm . a best - fit 3 rd order polynomial standard curve was developed for each plate ( r 2 ≧ 20 . 99 ), and these equations were used to calculate pge 2 concentrations for samples from each plate . hyaluronic acid in synovial fluid samples were digested with hyaluronidase as described above . gag concentration of synovial fluid was determined using a 1 , 9 - dmb spectrophotometric assay as described by chandrasekhar et al . ( 1987 ). samples were diluted 1 : 3 with dilution buffer and placed into a 96 - well microtitre plate . guanidine hydrochloride ( 275 g / l ) was added to each well followed immediately by addition of 150 μl dmb reagent . plates were incubated in the dark for 10 minutes , and absorbance was read on , a victor 3 microplate reader at 530 nm . sample absorbance was compared to that of a bovine chondroitin sulfate standard ( sigma , oakville on ). a best - fit linear standard curves was developed for each plate ( r 2 ≦ 0 . 99 ), and these equations were used to calculate gag concentrations for samples on each plate . nitrite ( no 2 — ), a stable oxidation product of no , was analyzed by the griess reaction ( fenton et al ., 2002 ). undiluted tcm samples were added to 96 well plates . sulfanilamide ( 0 . 01 g / ml ) and n -( 1 )- napthylethylene diamine hydrochloride ( 1 mg / ml ) dissolved in phosphoric acid ( 0 . 085 g / l ) was added to all wells , and absorbance was read within 5 minutes on a victor 3 microplate reader at 530 nm . sample absorbance was compared to a sodium nitrite standard . two - way repeated measures ( rm ) analysis of variance ( anova ) was used to detect differences between treatments . when a significant f - ratio was obtained , the holm sidak post - hoc test was used to identify differences between treatments . one - way rm anova was used to detect differences within treatments with respect to time . for blood and synovial fluid data , one - way comparisons of data were made against pre - and inj - 1 data , as each represented baseline for diet and il - 1 injections , respectively . data are presented as means ± sem . graphs for biochemistry and hematology data are scaled to physiological reference intervals unless otherwise stated . reference intervals are those published by the animal health laboratory , university of guelph ( http // www . labservices . uoguelph . ca / units / ahl / files / ahl - userguide . pdf ). con horses : there was no significant change in synovial fluid [ pge 2 ] in saline - injected joints at any time ( fig7 , a ). relative to pre - injection concentrations , [ pge 2 ) was significantly increased at inj - 2 - 2 ( 321 . 3 ± 161 . 8 pg / ml ; p = 0 . 04 ) in il - 1 - injected joints , at which time synovial fluid [ pge 2 ] was significantly higher in il - 1 - injected joints than in saline - injected joints ( p & lt ; 0 . 001 ). seq horses : data represent n = 5 , as one outlier horse was removed from the analysis . pge 2 did not change in saline - injected joints of seq horses . like con horses , there was a spike in [ pge 2 ] increased at inj - 2 - 2 ( 175 . 4 ± 89 . 2 pg / ml ) in il - 1 - injected joints of seq horses ( fig7 , b ). however , this increase was not significant when compared with pre - injection concentrations . pge 2 response to saline injection was not different in seq horses compared with con horses . there was no significant difference in pge 2 response to il - 1 injection compared with saline in seq horses . although mean [ pge 2 ] at inj - 2 - 2 in seq horses was approximately 55 % that of con horses , variability about the means resulted in no significant difference between diets . con horses : synovial fluid [ gag ] increased in saline - injected joints between inj - 1 ( 18 . 3 ± 6 . 8 μg / ml ) and day 1 ( 48 . 1 ± 9 . 6 μg / ml ) ( fig8 , a ). injection of il - 1 ( 10 ng ) caused a rapid and significant increase in synovial fluid [ gag ] between inj - 1 ( 24 . 5 ± 7 . 3 μg / ml ) and inj - 2 ( 77 . 6 ± 4 . 4 μg / ml ). synovial fluid [ gag ] remained significantly elevated in il - 1 - injected joints at inj - 2 - 2 ( 66 . 0 ± 9 . 6 μg / ml ) and day 1 ( 53 . 3 ± 11 . 4 μg / ml ) compared with pre - injection concentrations . the magnitude of increase in synovial fluid [ gag ] was significantly higher in il - 1 - injected joints than in saline - injected joints ( p = 0 . 003 ). seq horses : synovial fluid [ gag ] tended to increase ( p = 0 . 09 ) in both saline - and il - 1 - injected joints between pre ( saline : 29 . 3 ± 5 . 9 μg / ml ; il - 1 : 27 . 0 ± 10 . 8 μg / ml ) and inj - 1 ( saline : 85 . 5 ± 28 . 0 μg / ml ; il - 1 ; 83 . 2 ± 27 . 9 μg / ml ), suggesting an effect of diet on synovial fluid [ gag ] ( fig8 , b ). there was no change in synovial fluid [ gag ] in saline - or il - 1 - injected joints over the course of the experiment . there was no significant difference in synovial fluid [ gag ] of il - 1 - injected and saline - injected joints . synovial fluid [ gag ] in il - 1 - and saline - injected joints was significantly higher in seq horses than con horses ( p & lt ; 0 . 001 ). this difference . was mainly an effect of diet , and not an effect of il - 1 , as evidenced by the fact that the majority of the increase occurred prior to any il - 1 injection . con horses : synovial fluid [ no ] was low and variable over the course of the experiment in both saline - and il - 1 - injected joints . there was no significant effect of either saline or il - 1 injection on no levels in con horses over time ( data not shown ). the magnitude of synovial fluid [ no ] was not different between il - 1 - and saline - injected joints . seq horses : there was no change in synovial fluid [ no ] in il - 1 - or saline - injected joints at any time over the course of the experiment . there was no significant difference between il - 1 or saline at any time . there was no significant effect of diet on synovial fluid [ no ] in il - 1 - or saline - injected joints . con horses : pre - injection total cell count ( 0 . 61 ± 0 . 1 × 10 9 / l ) was significantly elevated by provision of exogenous il - 1 ( 10 ng ) at inj - 2 ( 40 . 17 ± 16 . 1 × 10 9 / l ). cell count was not further increased following the 2 nd il - 1 injection ( 100 ng ), but remained slightly ( but not significantly ) elevated through day 1 . inj - 1 celfcount in saline - injected joints ( 0 . 6 ± 0 . 2 × 10 9 / l ) increased mildly , reaching a maximum at day 1 ( 6 . 0 ± 2 . 6 × 10 9 / l ), but this increase was not significant . total cell counts of saline - and il - 1 injected joints were significantly different from each other at inj - 2 [ ie . 24 h after the 1 st il - 1 injection ( 10 ng )]. the increase in cell count was due mainly to an increase in the relative percentage of neutrophils . percent neutrophils significantly increased in both il - 1 - and saline - injected joints after the first injection . neutrophil counts significantly declined in both il - 1 - and saline - injected joints between day 1 and 3 without further increase for the remainder of the experiment . there was no difference in % neutrophils between il - 1 - and saline - injected joints ( data not shown ). seq horses : pre - injection total cell count ( 0 . 4 ± 0 . 03 × 10 9 / l ) was significantly elevated by provision of exogenous il - 1 ( 10 ng ) by inj - 2 ( 27 . 5 ± 8 . 7 × 10 9 / l ). cell count was not further increased by inj - 2 - 2 , but remained significantly elevated through day 1 . inj - 1 total cell count in saline - injected joints ( 0 . 4 ± 0 . 1 × 10 9 / l ) increased mildly , reaching a maximum at inj - 2 - 2 ( 4 . 0 ± 2 . 6 × 10 9 / l ), but this increase was not significant . total cell counts of saline - and il - 1 injected joints were significantly different from each other at inj - 2 ( ie . 24 h after the 1 st il - 1 injection of 10 ng ), inj - 2 - 2 ( ie . 8 h after the 2 nd il - 1 injection of 100 ng ), and day 1 ( ie . 24 h after the 2 nd il - 1 injection of 100 ng ). percent neutrophils significantly increased in both il - 1 - and saline - injected joints after the first injection . increase in neutrophil concentration of saline - injected joints may have been attributable to minor inflammation being caused by injection trauma . neutrophil counts (%) significantly declined in both il - 1 - and saline - injected joints between day 1 and 3 with a second significant spike on day 7 . there was no difference in % neutrophils between il - 1 - and saline - injected joints . there was no significant difference in the effect of seq and con diets on total cells counts or % neutrophils in il - 1 - or saline - injected joints . con horses : synovial fluid [ protein ] was significantly increased by injection of 10 ng il - 1 ( 20 ± 0 . 0 g / l to 39 . 4 ± 4 . 0 g / l ) ( fig9 , a ). [ protein ] was not further increased by injection of 100 ng il - 1 , and significantly declined 24 h after the 100 ng injection . injection of saline also resulted in a significant increase in [ protein ] immediately after the first injection , returning to baseline concentrations by day 1 ( 25 . 5 ± 1 . 5 g / l ). the magnitude of increase in [ protein ] over the course of the experiment was significantly higher in il - 1 - injected than saline - injected joints ( p = 0 . 01 ). seq horse &# 39 ; s : injection of 10 ng il - 1 resulted in a significant increase in synovial fluid protein on inj - 2 ( 38 . 7 ± 4 . 9 g / l ), inj - 2 - 2 ( 36 . 2 ± 4 . 4 g / l ), and day 1 ( 27 . 8 ± 3 . 8 g / l ) compared with inj - 1 ( 20 ± 0 g / l ) ( fig9 , b ). there was no further effect of the 2 nd il - 1 injection of 100 ng on [ protein ]. saline injection also resulted in a significant increase in [ protein ] on inj - 2 - am ( 27 . 5 ± 3 . 0 g / l ) and inj - 2 - pm ( 25 . 8 ± 2 . 5 g / l ) compared with inj - 1 ( 20 . 6 ± 0 . 6 g / l ). the magnitude of increase in synovial fluid [ protein ] was significantly higher in il - 1 - injected joints than in saline - injected joints ( p = 0 . 003 ). there was no significant difference in the effect of seq and con diets on synovia fluid [ protein ] in il - 1 - or saline injected joints . con horses : there was no significant change in circumference over time in il - 1 - or saline - injected joints , and there was no significant difference in joint circumference between il - 1 - and saline - injected joints ( fig1 , a ). seq horses : there was a significant increase in joint circumference in il - 1 - injected joints between inj - 1 ( 31 . 1 ± 0 . 2 cm ) and inj - 2 ( 31 . 9 ± 0 . 5 cm ) in seq horses ( fig1 , b ). joint circumference remained significantly elevated at inj - 2 - 2 ( 31 . 7 ± 0 . 4 cm ) before declining to pre - injection levels . exactly the same pattern was shown in the saline - injected joints of seq horses . joint circumference of il - 1 - injected joints was significantly lower in seq horses than con horses ( p & lt ; 0 . 001 ) this data shows a minimally invasive , reversible model of early stage articular inflammation that can be used to evaluate putative anti - inflammatory nutraceuticals . the double il - 1 injection protocol resulted in a statistically significant increase in pge 2 at 8 h after the 2 nd injection . none of the con horses were overtly lame at the walk or brief trot at any time during the experiment , despite mean peak synovial fluid [ pge 2 ] ( 498 pg / ml ) being commensurate with that associated with lameness in horses ( 488 pg / ml ; de grauw et al ., 2006 ). the increase in pge 2 was not accompanied by a concomitant increase in no . this provides a possible explanation as to why these horses were not lame , as transmission and perception of nociceptive pain occurs predominately as a result of combined effect of elevated pge 2 and no . con horses may have demonstrated a low - grade lameness had they been subjected to moderate exercise , but this was not undertaken due to the confounding effect of exercise on synovial fluid [ pge 2 ] ( van den boom et al ., 2005 ). the observed increase in synovial fluid [ pge 2 ] in con horses provides good evidence for a low - grade il - 1 - induced inflammation within the joint . we hypothesized that this increase would be blunted by dietary provision of an efficacious anti - inflammatory nutraceutical . trafficking of inflammatory cells and release of glycosaminoglycan into the synovial fluid were more sensitive to stimulation with il - 1 than production of pge 2 , as an increase in synovial fluid [ gag ] and [ neutrophils ] was observed 24 h after the initial 10 ng ill injection . synovial fluid [ protein ] was also elevated immediately after the 1 st il - 1 injection . these parameters were not further increased by provision of a higher il - 1 challenge . these responses are consistent with a ‘ pre - arthritic ’ inflammatory state ( adarichev et al ., 2006 ). genes turned on in the early stage of arthritis are predominately those associated with transcription of chemokines , cytokines ( notably , il - 1 ), and metalloproteinases , notably , mmp - 13 and mmp - 9 . chemokines are potent signals for inflammatory cell migration into the synovial space . as synoviocytes and endothelial cells of the synovial membrane become activated to express cell adhesion molecules and produce chemokines , neutrophil extravasation into the joint space greatly increases , as was observed in the studies described herein as a steep increase in synovial fluid [ neutrophils ]. cells of the synovial membrane also become more permeable to serum proteins ( middleton et al ., 2004 ) resulting in the observed rapid increase in synovial fluid [ protein ]. mmp - 13 ( yammani et al ., 2006 ) and mmp - 9 ( soder et al , 2006 ) are key degradative enzymes in articular cartilage , and the increase in il - 1 - induced synovial fluid [ gag ] observed in the current study support studies demonstrating substantial upregulation of genes encoding these enzymes in early arthritis ( adarichev et al ., 2006 ; kydd et al ., 2007 ). micro - array analysis of pre - arthritic cartilage in pg - stimulated mice revealed that genes encoding for phospholipase c 2 , the enzyme catalyzing release of arachidonic acid from nuclear membranes , was not elevated ( adarichev et al ., 2006 ). this may explain , at least in part , why pge 2 required ‘ a longer time course for elevation subsequent to il - 1 stimulation than cell migration and release of gags . intra - articular challenge with il - 1 did not result in a consistent increase in synovial fluid nitric oxide . il - 1 - induced nitric oxide has been frequently reported in cartilage explant models ( pearson et al ., 2007 ; petrov et al . 2005 ), cells taken from animal models of acute articular inflammation ( kumar et al ., 2006 ) and clinical cases of articular inflammation ( karatay et al ., 2005 ). this data provides support for evidence that genes encoding inducible nitric oxide synthase are not upregulated in early stage arthritis ( kydd et al ., 2007 ), which delays il - 1 - induced formation of nitric oxide . seq provided protection to il - 1 - stimulated joints as evidenced by : 1 ) no significant increase in synovial fluid [ pge 2 ]; 2 ) increased [ gag ] in the synovial fluid prior to il - 1 challenge , then preventing il - 1 - induced increase in gag ; and 3 ) limited effusion into the joint space subsequent to il - 1 challenge . as part of the diet for 2 weeks prior to an intra - articular il - 1 challenge , seq prevented significant elevation in il - 1 - induced pge 2 . similar to con horses , pge 2 response to il - 1 in seq horses peaked at 8 h after the second il - 1 injection , but the peak was lower , and did not result in statistically significant changes over time or significant differences between il - 1 and saline injection . this shows that seq reduces inflammation and pain associated with elevated pge 2 in horses with early stage arthritis , and implies that feeding seq to horses prior to articular damage may impede progression of the disease to a more advanced stage . the observed increase in synovial fluid [ gag ] of seq horses in both saline - and il - 1 - injected joints between pre and inj - 1 — ie . before inflammatory challenge — provides evidence for the post - absorptive accumulation of dietary gags within the synovial space . the effectiveness of seq in preventing biochemical indicators of early - stage arthritis results from a synergistic effect of its four ingredients . published reports have reported significant improvement in arthritic signs in dogs provided with dietary nzglm ( pollard et al ., 2006 ), and significant protection by glucosamine and chondroitin — the major bioactive constituents of sc — of cartilage explants against degradation by il - 1 ( dechant et al ., 2005 ). however , the in vitro pge 2 - inhibitory effect of seq is greater than that of any of its four constituents alone , per gram of product ( pearson et al . unpublished ), suggesting a level of synergism between the ingredients . oil from the seeds of biota orientalis was fractionated using an agilent 1200 preparative hplc equipped with a diode array detector and an automated fraction collector . the column used was an agilent prep c18 . 10 μm ( 30 × 250 mm ) with the following gradient at a flow rate of 20 ml / minute with a 900 μl injection of constituent 4 . 0 - 5 minutes 80 % water 20 % acetonitrile . 5 - 7 minutes gradient change to 10 % water 90 % acetonitrile , 7 - 25 minutes isocratic 10 % water 90 % acetonitrile . fraction detection was achieved at 254 nm . the mass spectrometry detection was performed on an agilent 6210 msd time of flight mass spectrometry in both positive and negative ion mode . the following electrospray ionization conditions were used , drying gas : nitrogen ( 7 ml min - 1 , 350 ° c . ); nebuliser gas : nitrogen ( 15 psi ); capillary voltage : 4 . 0 kv ; vaporization temperature : 350 ° c . and cone voltage : 60v fig1 shows the chromatographic spectrum of the oil , and various fractions were collected and numbered as shown . to study the anti - inflammatory activities , assays fr 1 , fr i , fr v and fr vi were selected and tested at a concentration of ≦ 64 μg / ml . the assays carried out to measure the 1 ) nitric oxide ( no ) levels , 2 ) prostaglandin pge2 levels , 3 ) prostaglandin pgf2α levels . nhac cells at passage 3 , were stimulated first with proinflammatory cytokine il - 1β at a predetermined concentration 10 ng / ml overnight , nhac cells were then treated with fractions in the presence of 1l - 1β 10 ng / ml for 24 hours and cell culture supernatant was collected to measure no , pge2 and pgf2α levels . griess reagent kit for nitrite determination ( molecular probes , invitrogen ) was used as per kit instructions . for estimation of pgs , high sensitivity pge2 & amp ; pgf2α eia kits ( assay designs inc .) were used . as shown in fig1 , fractions 1 ( fr 1 ), fr i , and fr v reduced the no levels ( highly significant ) in a dose dependent manner . fr1 was found to be the most effective among all the four fractions with fr vi the least effective , although still showing some effect . the non steroidal anti inflammatory drug indomethacin used as a positive control significantly reduced the il - 1β induced pge2 levels . all the four fractions had no effect on these levels at any of the concentrations tested ( fig1 & amp ; 17 ). indomethacin significantly reduced the il - 1β induced pgf2α levels . fr 1 showed no effect at all on the pgf2α levels , while fr i , fr v and fr vi reduced these levels , in a dose dependent manner ( 64 - 32 μg / ml ) ( fig1 & amp ; 19 ). the effectiveness of the biota oil extract fractions has until now not been known . the use of the compounds of f1 . 1 - 1 . 4 either separately or as a mixture with one or more of the other fractions provides for a remarkable improvement in the treatment of conditions , such as osteoarthritis . any improvement may be made in part or all of the method steps and systems components . all references , including publications , patent applications , and patents , cited herein are hereby incorporated by reference . the use of any and all examples , or exemplary language ( e . g ., “ such as ”) provided herein , is intended to illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed . any statement herein as to the nature or benefits of the invention or of the preferred embodiments is not intended to be limiting , and the appended claims should not be deemed to be limited by such statements . more generally , no language in the specification should be construed as indicating any non - claimed element as being essential to the practice of the invention . this invention includes all modifications and equivalents of the subject matter recited as permitted by applicable law . moreover , any combination of the above - described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contraindicated by context . adarichev v a , vermes c , hanyecz a , ludanyi k , tunyogi - csapo m , finnegan a , mikecz k , giant t t . 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