inflammation is considered the main response of the body evoked to deal with injuries and its hallmarks include swelling , redness , pain , and fever . several mediators regulate the events of acute inflammation , influence vascular changes , and provoke inflammatory cell recruitment [ 13 ] . the inflammatory response is a complex self - limiting process precisely regulated to prevent extensive damage of the host . when the self - limiting nature of this protective mechanism is inappropriately regulated , it is transformed to a detrimental chronic state of inflammation . several diseases are associated with chronic inflammation , including osteoarthritis , atherosclerosis , diabetes , obesity , crohn 's disease , and cancer [ 13 ] . although the today used steroidal anti - inflammatory drugs ( said ) and nonsteroidal anti - inflammatory drugs ( nsaids ) effectively manage the acute inflammatory reaction , in chronic inflammatory states the long - term treatment is followed by severe adverse effects . recently , there has been interest in the potential of flavonoids to modulate the chronic inflammatory reaction . flavonoids are major constituents of fruits , vegetables , and beverages , such as wine , tea , cocoa , and fruit juices . flavonoids have a similar structure consisting of two aromatic rings ( a and b ) , which are bound together by three carbon atoms , forming an oxygenated heterocycle ( ring c ; figure 1 ) . according to changes in the chemical structure , flavonoids are divided into seven classes : flavonols , flavones , flavanones , flavanonols , flavanols , anthocyanidins , and isoflavones . the flavanols , also referred to as flavan-3-ols , are mainly present in green and black tea , red wine , chocolate , and fruits such as apples , grapes , and strawberries . typical dietary flavanols include catechin , epicatechin , epigallocatechin ( egc ) , and epigallocatechin gallate ( egcg ) . flavones , such as apigenin and luteolin , are found in parsley , chives , artichoke , and celery . dietary flavanones include naringenin , hesperetin , and taxifolin and are found mainly in citrus fruit and tomatoes . finally , isoflavones such as genistein and daidzein are a subclass of the flavonoids family found in soy and soy products . they have a large structural variability and more than 600 isoflavones have been identified to date and are classified according to oxidation level of the central pyran ring ( figure 1 ) . acacia catechu , also called black catechu , is a traditional medicinal plant commonly used in indian subcontinent and southeast asia , with antipyretic , antidiarrheal , hypoglycaemic , and hepatoprotective activities [ 5 , 6 ] . the major chemical components in an acacia catechu extract are catechin and epicatechin-3-o - gallate . catechin reduces inflammation and blunts the production of proinflammatory cytokines , such as interleukin-1 ( il-1 ) and tumor necrosis factor- ( tnf- ) , as demonstrated in rats with experimental arthritis . scutellaria baicalensis , a traditional chinese herbal medicine , also called chinese skullcap , has been used to treat respiratory inflammatory diseases [ 812 ] , viral infections [ 1315 ] , cardiovascular diseases [ 1618 ] , and cancer [ 19 , 20 ] . baicalin , extracted by scutellaria baicalensis , inhibits cyclooxygenase cox-1 and cox-2 peroxidase and 5-lipoxygenase ( 5-lox ) enzyme activities , decreases production of proinflammatory eicosanoids , and attenuates edema in an in vivo model of inflammation . the anti - inflammatory activity of baicalin was also associated with a binding inhibition of chemokines , such as macrophage inflammatory protein ( mip)-1 , monocyte chemotactic protein-2 ( mcp-2 ) , causing a reduced capacity of the chemokines to induce cell migration . the flavonoids , baicalin and catechin , modulate the activities of arachidonic acid , metabolizing enzymes involved in this pathway , and inos ( no producing enzyme ) . flavocoxid is a mixture of the flavonoid molecules catechin , from acacia catechu , and baicalin , extracted from scutellaria baicalensis , concentrated to greater than 90% purity ( figure 2 ) . thanks to its components flavocoxid , in addition to anti - inflammatory properties , may also act as an antioxidant , reducing reactive oxygen species including hydroxyl radical , superoxide anion radical , and hydrogen peroxide . the combined effect of the 2 flavonoids is greater than the isolated molecules , as shown in a recent paper . as a consequence of nf-b modulation , flavocoxid reduces cox-2 , 5-lox , inos , and tnf production , and it also blunts the formation of cox-2 , 5-lox , and inos metabolites , as pge2 , ltb4 , and nitrates [ 2224 ] . these effects provide a rationale for the use of a dual inhibitor in acute and chronic inflammatory conditions . flavocoxid , marketed as limbrel in the usa , is a usfda - regulated prescription , and it is expected to have significant therapeutic efficacy in the managing of chronic inflammation . this review will focus on the preclinical pharmacology , toxicology , and clinical pharmacology of this new attractive compound . flavocoxid is prepared from roots of scutellaria baicalensis and acacia catechu ( us patent number 7,514,469 ) . the roots of scutellaria baicalensis are extracted with 70% ethanol and then recrystallized with an ethanol / water solvent . the scutellaria baicalensis extract contains baicalin as the major component and additional minor free - b - ring flavonoids . in the roots of acacia catechu , ( + ) -catechin is the major component at a content of 80% . analysis of the extracts is carried out separately by high performance liquid chromatography with photodiode array detector ( hplc / pda ) and liquid chromatography - mass spectrometry ( lc / ms ) and indicates a major compound , baicalin , from scutellaria baicalensis extract and ( + ) -catechin from acacia catechu extract by comparison with known standards . the presence of these compounds is then confirmed by carbon nuclear magnetic resonance ( c - nmr ) and proton nuclear magnetic resonance ( h - nmr ) analysis , respectively . the final flavocoxid formulation ( figure 2 ) is a mixture of > 90% purified baicalin and catechin with the remainder being excipient ( 5 - 6% ) and water ( 3% ) . both flavonoids are detected using uv detector at 275  nm and identified based on retention time by comparison with known flavonoids standards . these ingredients are generally recognized as safe ( gras ) . for an ingredient to be recognized as gras by the us food and drug administration ( fda ) , it requires technical demonstration of nontoxicity and safety , general recognition of safety through widespread usage , and agreement of that safety by experts in the field . a series of in vitro and in vivo experiments have been carried out in order to dissect out the exact mechanism of action of flavocoxid . first of all , flavocoxid was tested in peritoneal macrophages ( m ) stimulated with lipopolysaccharide ( lps ) to investigate a possible effect on phospholipase a2 ( pla2 ) activity . flavocoxid did not significantly modify cell viability at 200 and 500  g / ml , but it markedly inhibited pla2 activity ( ic50 = 60  g / ml ) at doses of 50 , 100 , 200 , and 500  g / ml . this finding indicates that flavocoxid modulates the generation of aa from membrane phospholipids caused by tissue damage during chronic inflammation . the effects of flavocoxid on cox-1 and cox-2 enzyme activities were also investigated in dedicated in vitro enzyme assays . cox proteins have two different enzymatic moieties for aa metabolism : the cyclooxygenase ( co ) one and the peroxidase one ( po ) . the co activity converts aa to pgg2 and the po activity transforms pgg into pgh2 . finally , cell synthases and isomerases convert pgh2 to thromboxanes ( txb ) , prostaglandins ( pg ) , and prostacyclin ( pgi ) . experiments were carried out to investigate the specific inhibitor effects of flavocoxid on co and po enzyme moieties of either cox-1 or cox-2 . the compound had no significant anti - co cox-2 activity up to 50  g / ml . in addition , flavocoxid showed co cox-1 ic50 of 25  g / ml , while indomethacin has a co cox-1 ic50 of 0.012  g / ml , thus suggesting that flavocoxid has little anti - co activity on both cox enzymes compared to well - known anti - inflammatory agents . imbalances in cox-2 versus cox-1 inhibition caused by selective cox-2 lead to production of several aa metabolites responsible for an enhanced risk of edema , hypertension , and myocardial infarction . flavocoxid produced a balanced inhibition of both cox-1 and cox-2 po activities , with ic50 of 12.3 and 11.3  g / ml , respectively . this clearly indicates that flavocoxid exerts its activity via modulation of the po activity of these enzymes . currently available nsaids and cox-2 inhibitors do not influence the 5-lox pathway and therefore do not block the production of the dangerous leukotrienes ( lts ) that cause vasoconstriction and leukocyte attraction and accumulation . furthermore cox-1 and cox-2 blockade by nsaids or selective cox-2 inhibitors causes a shunting of the aa metabolism towards the 5-lox pathway , thus inducing an overproduction of these fatty acid mediators which can cause multiple organ damage . enhanced levels of lts are also measured in a variety of pathological conditions such as asthma , gastric ulceration , renal insufficiency , and cardiovascular complications [ 27 , 28 ] . flavocoxid was incubated along with purified 5-lox enzyme in the presence of an oxygen sensing chromagen in vitro to study the formation of unstable hydroperoxyeicosatetraenoic acids ( hpetes ) intermediates in the synthesis of lts . phenidone , a well - known 5-lox inhibitor used as a positive control in theses assays , had an ic50 of 1.3  g / ml . no other nsaids or selective cox-2 inhibitors , including rofecoxib , valdecoxib , diclofenac , meloxicam , and aspirin , displayed an anti-5-lox activity . all these findings , taken together , indicate that flavocoxid also reduces the production of ltbs from 5-lox and may avoid the deleterious accumulation of these lipid mediators caused by the nsaids - induced 5-lox shunt . reactive oxygen species ( ros ) may react with exaggerated aa levels leading to the production of f2-isoprostanes and 4-hydroxynonenal ( hne ) together with enhanced malondialdehyde levels . all these markers of oxidative stress are elevated during chronic inflammation and contribute to the pathological cascade leading to organ damage and dysfunction . therefore , it is relevant for an anti - inflammatory agent to possess an antioxidant effect . experiments were carried out to analyze this issue , and the in vitro antioxidant activity of flavocoxid was evaluated using oxygen radical absorbance capacity ( orac ) procedures and was compared with that of well - known antioxidants such as vitamin c and vitamin e. the orac analysis provides a measure of the scavenging capacity of antioxidants against the peroxyl radical . trolox , a water - soluble vitamin e analog , is used as the calibration standard , and the orac result is expressed as molte / g dry weight . the oractotal for flavocoxid ( 3719  molte / g ) was significantly greater than that of either vitamin c ( 2000  molte / g ) or vitamin e ( 1100  molte / g ) . flavocoxid showed also high values for the ferric reducing / antioxidant power ( frap ) , the peroxynitrite radical averting capacity ( norac ) , and the trolox equivalent antioxidant capacity ( teac ) , thus clearly showing that this compound exerts a strong antioxidant activity . the effects of flavocoxid on gene and protein expression of inflammatory markers were studied in rat peritoneal macrophages stimulated with salmonella enteritidis lps . in fact it is worthy of interest to identify for an anti - inflammatory agent a mechanism of action at the level of inflammatory gene and protein expression . by contrast lps stimulation of peritoneal macrophages resulted in a marked increase in both cox-2 and 5-lox expression . flavocoxid attenuated , in a concentration dependent manner , the increase in cox-2 and 5-lox expression . together with lps - induced cox-2 and 5-lox activation , the generation of pge2 and ltb4 was markedly augmented . in addition , lps - primed macrophages had an enhanced mrna expression for inos and augmented nitrate content ; flavocoxid significantly blunted , in a concentration dependent manner , the increase in inos and nitrate production . these results [ 22 , 24 , 29 ] suggest that flavocoxid inhibits cox-2 , 5-lox , and inos gene activation and abrogate the biosynthesis of related inflammatory mediators . among these mediators an important role is played by tnf- , a pleiotropic proinflammatory cytokine , markedly induced in peritoneal macrophages stimulated with lps . flavocoxid caused a significant and concentration dependent reduction in the levels of tnf- mrna and in the formation of the mature protein . nuclear factor kappa - b ( nf-b ) plays a prominent role in the inflammatory cascade ; it is an important transcription factor complex that regulates the expression of several genes involved in immune and inflammatory responses during chronic human diseases [ 32 , 33 ] . in unstimulated cells , nf-b is constitutively localized in the cytosol as a heterodimer by physical association with an inhibitory protein , called ib. following activation , the nf-b heterodimer is rapidly translocated to the nucleus where it activates the transcription of target genes , including the genes encoding for proinflammatory cytokines , adhesion molecules , chemokines , and inducible enzymes ( such as cox-2 , 5-lox , and inos ) . flavocoxid was shown to reduce ib loss from the cytoplasm and to blunt nf-b binding to dna in lps - stimulated macrophages . therefore flavocoxid , acts at gene and protein expression level through nf-b activity inhibition , blocking the auto - amplifying loop during the inflammatory response . flavocoxid was studied in animal experimental models of inflammation aimed to test its efficacy . in a first experiment , collagen - induced arthritis ( cia ) was induced in dba/1 mice by an intradermal injection of an emulsion containing bovine type ii collagen in complete freund 's adjuvant . cia animals were then randomized to receive vehicle or flavocoxid ( 20  mg / kg ) and the treatment lasted 45 days ( figure 3 , data on file ) . flavocoxid reduced pge2 and ltb4 levels , significantly ameliorated the clinical signs of arthritis , improved the histological damage , decreased the cartilage expression and the circulating levels of several markers of severity disease including tnf- , il-6 , high mobility group box-1 ( hmbg-1 ) , and also caused an enhanced expression of the anti - inflammatory cytokine il-10 . interestingly , flavocoxid positively modulated the balance between receptor activator of nuclear factor-b ligand ( rankl ) and osteoprotegerin ( opg ) , a cytokine system involved in bone and cartilage remodeling . collectively these experimental findings demonstrate that flavocoxid is a valuable therapeutic agent in the treatment of inflammatory conditions , including arthritis and osteoarthritis . the dual inhibitor was also tested in acute inflammatory diseases such as acute pancreatitis , an autodigestive inflammatory disease , that causes acinar cell damage and finally culminates in hemorrhagic necrosis of the pancreas and eventually multiple organ failure . a large body of evidence suggests that upregulation of inflammatory mediators , including cox-2 , 5-lox , cytokines , and chemokines , orchestrates this pathological process . acute pancreatitis can be induced in rats by injection of cerulein , a secretagogue agent . flavocoxid was investigated for its effects in cerulein - induced pancreatitis   at a dose of 20  mg / kg ; it inhibited cox-2 and 5-lox expression and reduced serum levels of lipase and amylase and the degree of pancreatic edema . administration of flavocoxid also blunted the increased pancreatic tnf- mrna expression , serum ltb4 and pge2 levels , and protected against histological damage in terms of vacuolization and leukocyte infiltration . these interesting findings may provide a potential therapeutic approach to the treatment of patients at high risk of developing this life - threatening condition . flavocoxid was also tested in a degenerative chronic disease , duchenne muscle dystrophy ( dmd ) , a progressive muscle - wasting disease leading to death , usually in early adulthood . the disease results from absence of the protein dystrophin , which is an essential component of the dystrophin - glycoprotein complex that maintains membrane integrity of muscle fibers by linking cytoskeleton to extracellular matrix . muscle degeneration in muscle dystrophy is exacerbated by the endogenous inflammatory response and increased oxidative stress , and nf-b plays a pivotal role in orchestrating this inflammatory cascade [ 38 , 39 ] . the effects of flavocoxid were studied in a comparison study with methylprednisolone , the gold standard treatment for dmd patients , using the mdx mice , the murine model of dmd . five - week - old mdx mice were treated for 5 weeks with flavocoxid , methylprednisolone , or vehicle . flavocoxid was more effective than methylprednisolone in ameliorating functional properties both in vivo and in vitro ; in reducing serum creatine kinase ( ck ) , a marker of muscle necrosis ; in blunting the expression of oxidative stress markers and inflammatory mediators . furthermore more efficiently than methylprednisolone , flavocoxid inhibited nf-b and mitogen - activated protein kinases ( mapks ) signal pathways , decreased muscle necrosis , and augmented muscle regeneration . this experiment suggests that flavocoxid counteracts the chronic inflammatory cascade that contributes to muscle necrosis and degeneration in dmd showing a degree of activity higher than that of methylprednisolone . since methylprednisolone possesses also important side effects , flavocoxid with a better safety - efficacy profile might represent a valuable alternative in the treatment of dmd . however this hypothesis deserves to be confirmed in a clinical setting . in another experimental model of chronic inflammation , benign prostatic hyperplasia ( bph ) , we tested the effects of flavocoxid since cox and 5-lox are significantly elevated in the overgrowing prostate . therefore a  dual inhibitor  of the cox and 5-lox enzymes might be of benefit in this disease . rats were treated , daily , with testosterone propionate ( 3  mg / kg / sc ) or its vehicle for 14 days . testosterone administered animals were randomized to receive vehicle ( 1  ml / kg , ip ) or flavocoxid ( 20  mg / kg , ip ) for 14 days . flavocoxid reduced prostate weight and hyperplasia , blunted the augmented expression of cox-2 and 5-lox as well as the increased production of pge2 and ltb4 , enhanced the proapoptotic bax and caspase-9 , and decreased the antiapoptotic bcl-2 mrna . flavocoxid reduced also the epidermal growth factor ( egf ) and vascular endothelial growth factor ( vegf ) expression . the data obtained from this study would indicate that a  dual inhibitor  of the cox and 5-lox enzymes , such as flavocoxid , might represent a rationale therapeutic approach to reduce benign prostate growth . a toxicological testing of the relative pure combination of baicalin and ( + ) -catechin has been performed in vitro and in experimental animals [ 41 , 42 ] . thp-1 cells , a human immortalized monocyte cell line , were used to assess cytotoxicity in an ldh assay . when cells were grown to confluent monolayer and then exposed to increasing concentration of several nsaids , celecoxib , and the baicalin/(+)-catechin combination , only indomethacin and celecoxib showed significant cytotoxicity above 50  g / ml . flavocoxid showed low cytotoxicity at the highest test concentration ( 100  g / ml ) . acute ( 2000  mg / kg / day for 14 consecutive days ) and subchronic ( 50 , 250 , and 500  mg / kg / day ) toxicity study in mice revealed no abnormalities in any toxicological end points examined including animal body weight , gross organ pathology and tissue histology , and blood chemistry or serology . flavocoxid was also administered in fisher 344 rats , a model for gastric toxicity of nsaids , and showed no sign of ulceration . the baicalin/(+)-catechin combination was tested at a 10  m concentration in a liver microsomal assay with endoplasmic reticulum fractions using spectrophotometric quantization of 7-benzyloxy-4-(trifluoromethyl)-coumarin as the substrate for cyp450 profiling . flavocoxid showed only moderate inhibition of cyp1a2 ( 23% ) at a 10  m concentration and low inhibition for all other cyp isoforms ( 11/16% ) . an additional 90-day oral toxicity study was carried out in hsd  :  sd rats to determine the potential of the baicalin/(+)-catechin combination to produce gastric toxicity . flavocoxid was administered at the dose levels of 250 , 500 , and 1000  mg / kg / day . there were no flavocoxid - related adverse events , including mortality , changes in body weight and food consumption , neurological effects ( assessed by the functional observational battery and motor activity ) , organ weight changes , or histopathological alterations . this study identified a dose of 1000  mg / kg / day / as the no - observed - adverse - effect level ( noael ) . a study was designed to compare the effectiveness and safety of flavocoxid to naproxen in subjects with moderate to severe osteoarthritis ( oa ) of the knee [ 43 , 44 ] . this was a randomized , multicenter , and double - blind study involving 220 subjects with oa of the knee that were randomized to receive either flavocoxid ( 500  mg twice daily ) or naproxen ( 500  mg twice daily for 12 weeks ) . primary outcome measures included the western ontario and mcmaster universities osteoarthritis index ( womac ) and subscales and timed walk . more than 90% of the subjects in both groups had significant reduction in the signs and symptoms of oa . no statistical significant difference in efficacy between flavocoxid and naproxen was observed [ 43 , 44 ] . flavocoxid treated patients had also significantly fewer upper gastrointestinal , renal , and respiratory adverse events ( aes ) . these results indicate that flavocoxid is effective as naproxen in the management of oa of the knee and shows a better safety profile than naproxen . an additional study in healthy volunteers found that flavocoxid does not affect the primary or extrinsic pathway of hemostasis and , by not inhibiting the anticoagulation effects of aspirin , may have utility in cardiovascular patients with chronic inflammation . an open - label , postmarketing study ( goal : gauging osteoarthritis [ oa ] with limber ) was performed to determine the overall efficacy and gastrointestinal tolerability of flavocoxid . a total of 1067 patients at 41 rheumatology practices were enrolled and prescribed flavocoxid 500  mg b.i.d . for 60 days . the physician global assessment of disease ( pgad ) visual analog scale ( vas ) was used as a global measure to assess the signs and the symptoms of oa including joint discomfort , functional stiffness , functional mobility , and quality of life . furthermore both overall tolerability and upper gi tolerability were assessed by individual questions scored on a 5-part likert scale . physicians were also asked to monitor any interruption in or cessation of use of flavocoxid due to the gi symptom as well as changes in the use of gastroprotective medications . a close monitoring of adverse events ( aes ) was also carried out . in the 1005 patients who completed all the follow - up visits the most important improvement was observed in patients with moderate to severe oa and in those subjects that were historically nonresponders to nsaids . a low incidence of o aes was also observed with a good overall and gi tolerability . in addition the use of flavocoxid resulted in a > 30% reduction in or cessation of the gastroprotective medications including proton pump inhibitors or histamine-2 receptor antagonists . overall this study , even if it was open label and not rigorously controlled , shows that flavocoxid possesses a significant efficacy in the management of oa with a good safety profile . steroidal anti - inflammatory drugs and nonsteroidal anti - inflammatory drugs are effective in the management of the acute inflammatory reaction , but they do not influence successfully chronic inflammatory states and possess severe adverse effects . flavocoxid is a mixture of the flavonoids molecules catechin , from acacia catechu , and baicalin , extracted from scutellaria baicalensis , concentrated to greater than 90% purity . flavocoxid hind pla2 causes a balanced inhibition of the cox-1 and cox-2 peroxidase moieties and decreases the generation of ltbs from 5-lox , avoiding the deleterious accumulation of these lipid mediators caused by the nsaids - induced 5-lox shunt . it has also a strong antioxidant activity , and through nf-b activity inhibition blocks the amplifying loop of the inflammatory response and acts at the level of gene and protein expression ( figure 4 ) . it exerts beneficial effects in several experimental models of inflammation . in vitro toxicity testing and acute and subchronic toxicity animal studies indicate for flavocoxid an optimal preclinical safety profile . finally , clinical trials and a postmarketing study show that flavocoxid has a significant efficacy in management of oa and a good overall and gi tolerability . flavocoxid may therefore provide a potential therapeutic approach to the treatment of acute and chronic inflammatory conditions .
flavonoids , from scutellaria baicalensis ( chinese skullcap ) and acacia catechu ( black catechu ) , have been shown to exert a variety of therapeutic effects , including anti - inflammatory , antiviral , antibacterial , and anticancer activities . 
 flavocoxid is a mixed extract containing baicalin and catechin and it acts as a dual balanced inhibitor of cyclooxygenase-1 ( cox-1 ) and cox-2 peroxidase enzyme activities with a significant inhibition of 5-lipoxygenase ( 5-lox ) enzyme activity in vitro . 
 flavocoxid downregulates gene or protein expression of several inflammatory markers and exerts also strong antioxidant activity in several experimental models . 
 controlled clinical trials and a postmarketing study have clearly shown that flavocoxid is as effective as naproxen in managing the signs and symptoms of osteoarthritis of the knee and it has better upper gastrointestinal , renal , and respiratory safety profile than naproxen . 
 flavocoxid may therefore provide a potential therapeutic approach to the treatment of chronic inflammatory conditions .