human rheumatoid arthritis ( ra ) is an autoimmune disease associated with painful joints that affects approximately 1% of the worldwide population , for which there is no effective cure available [ 1 , 2 ] . it arises from chronic joint inflammation , which causes structural damage to cartilage , bone , and ligaments . rheumatoid synovial tissue is central to the pathogenesis of ra , as it becomes hyperplastic as a result of the accumulation and proliferation of inflammatory cells , including b cells , t cells , macrophages , and synovial fibroblasts . infiltrating immune cells produce and release a variety of cytokines , predominantly tumor necrosis factor ( tnf)- and interleukin ( il)-1 , which attract and activate other inflammatory cells , propagating a vicious inflammatory cycle that leads to bone damage . these cytokines also increase the production of matrix metalloproteinases ( mmps ) , enzymes that can degrade all components of the extracellular matrix , leading to destruction of cartilage . ra is widely treated with nonsteroidal anti - inflammatory drugs , immunosuppressive disease - modifying anti - rheumatic drugs and corticosteroids , which normalize the immune system and reduce the expression of inflammatory mediators [ 6 , 7 ] . however , these drugs are undesirable for long - term treatment due to low efficacy , delayed onset of action , long - term side effects and toxicity . therapeutic targeting of cytokines in ra using tnf- and il-1 antagonists has substantial efficacy , but this strategy is expensive and has many side effects , including hypersensitivity to medications and infections [ 8 , 9 ] . therefore , it is necessary to develop preventive and therapeutic agents that have minimal toxicity , and oriental natural medicinal plants with anti - inflammatory activities are a useful resource to obtain novel antiarthritic agents . licorice is an esteemed crude drug that originates from the dried roots of several glycyrrhiza species . chinese traditional medicine practitioners have used licorice to treat various ailments ranging from tuberculosis to peptic ulcers , and licorice has been employed as a flavoring and sweetening agent as well as a demulcent and expectorant in western countries . the roasted form of licorice has been reported to possess anti - allergic , neuroprotective , antioxidative , and anti - inflammatory activities . in the present study , we found that roasted licorice extract ( rle ) was more potent than unroasted licorice extract ( le ) in inhibiting ear edema in mice treated with phorbol ester , which induces acute inflammation due to the increased activity of tnf- and il-1 . furthermore , we investigated the antiarthritic effects of rle in a murine collagen - induced arthritis ( cia ) model that has been widely accepted as an appropriate animal model of human rheumatoid arthritis . bovine collagen type ii ( cii ) , complete freund 's adjuvant ( cfa ) and incomplete freund 's adjuvant ( ifa ) were obtained from chondrex ( redmond , wa ) . 12-o - tetradecanoylphorbol-13-acetate ( tpa ) , dimethyl sulfoxide ( dmso ) , lipopolysaccharide ( lps ) , 1,1,3,3-tetramethoxypropane , 5,5-dithiobis ( 2-nitrobenzoic acid ) ( dtnb ) , and 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole ( purpald ) were purchased from sigma - aldrich chemical . female icr mice ( 5 weeks old ) and male dba/1j mice ( 79 weeks old ) were obtained from central laboratory animal ( seoul , korea ) and the jackson laboratory ( bar harbor , me ) , respectively . all mice were permitted free access to a normal standard chow diet and tap water , and were housed at 2022c , with a relative humidity of 55  5% and a 12-h light - dark cycle . animal studies were conducted in accordance with the guidelines for the use and care of animals of yonsei university ( seoul , korea ) . the right ear of each icr mouse ( 5 per group ) was topically treated with le or rle ( 1.0 and 2.0  mg / ear ) in 50  l vehicle ( dmso - acetone : 1585 , v / v ) 30 minutes prior to the application of 5  nmol tpa in 50  l vehicle . the left ears were treated with vehicle alone , and control mice received only vehicle on both ears . 4 hours later , right and left ear punches 6  mm in diameter were taken from each mouse , and then weighed . edema was represented as the increase in weight of the right ear punch over that of the left . dba/1j mice were intradermally injected with 100  g cii in cfa ( 1  :  1 , w / v ) to the tail base , and boosted with an intradermal injection of 100  g cii in ifa ( 1  :  1 , w / v ) on day 21 . cia mice were orally administered the vehicle ( pbs containing 1% dmso ) , le or rle at 10  mg / kg once daily from day 25 , when the first clinical signs of arthritis were observed , to day 45 . the development of arthritis was evaluated by macroscopic scoring as follows : 0 , normal ; 1 , swelling and/or redness in only one joint ; 2 , swelling and/or redness in more than one joint ; 3 , swelling and/or redness in the entire paw ; and 4 , severe swelling of the entire paw with deformity and/or ankylosis . the macroscopic arthritis score of each mouse was presented as the sum of each score of the four limbs , with the maximum score being 16 . paw swelling was assessed by measuring the thickness of the two hind paws with a digital caliper every other day from day 25 . on day 45 , all mice were sacrificed after serum collection . hind paw and knee joints , spleens , livers and kidneys were removed from the control and cia mice . the joint tissue sections were prepared and stained with hematoxylin and eosin after routine fixation , decalcification , and paraffin embedding . for immunohistochemical analysis , the sections were sequentially incubated with a specific primary antibody against mmp-3 ( santa cruz biotechnology , santa cruz , ca ) , and the appropriate horseradish peroxidase - linked secondary antibody . spleens were washed with cold pbs and dissociated into single cell suspensions using cell strainers . cells were cultured with rpmi 1640 medium containing 50  m 2-mercaptoethanol , 1% antibiotic - antimycotic mixture and 10% fbs . single - cell suspensions ( 2  10  cells / well ) were stimulated with 50  g / ml denatured - cii or 5  g / ml lps for 48 hours at 37c in a 5% co2-humidified incubator . tnf- and il-1 levels in the serum and in the conditioned medium were assayed using commercially available elisa kits ( r&d systems , minneapolis , mn ) . spleen single - cell suspensions ( 2  10  cells / well ) were stimulated with 50  g / ml denatured - cii for 72 hours at 37c in a 5% co2 humidified incubator . cell proliferation was measured using a brdu cell proliferation elisa kit ( roche diagnostics , mannheim , germany ) . lipid peroxidation , reduced glutathione ( gsh ) level and catalase activity in the liver and kidney homogenates were measured as described previously . protein concentrations were measured using the bca protein assay kit ( pierce , rockford , il ) . malondialdehyde ( mda ) levels were measured in form of thiobarbituric acid reactive substances at 532  nm . a standard curve was established using 1,1,3,3-tetramethoxypropane , and the mda level was expressed as nmol / mg protein . gsh levels were estimated at 412  nm colorimetrically after its complex formation with dtnb , and expressed as nmol / mg protein . the determination of catalase activity was based on the reaction of the enzyme with methanol in an optimal concentration of hydrogen peroxide . the formaldehyde produced was measured with purpald at 540  nm , and catalase activity was expressed as units / mg protein . one unit ( u ) of catalase was defined as the amount of enzyme that results in the formation of 1  nmol of formaldehyde per minutes at 25c . data are expressed as mean  standard error ( se ) , and analyzed via one - way anova with multiple comparisons , followed by tukey test . the present study is the first report to determine and compare the antiarthritic activities and underlying mechanism of le and rle in the cia mouse model of human ra . licorice is used in alternative medicine to treat individuals with gastric ulcers , bronchitis , arthritis , adrenal insufficiency and allergies [ 10 , 20 ] . it contains diverse chemical components including glycyrrhizin and its aglycone , as well as species - specific flavonoids such as licochalcone a , liquiritin , isoliquiritin and their corresponding aglycons [ 10 , 21 , 22 ] . recent studies demonstrated that rle showed more potent anti - allergic , anti - diabetic , and anti - inflammatory effects than le [ 13 , 20 , 26 ] , and had higher glycyrrhetinic acid and lower glycyrrhizin content than le . we have confirmed , using high - performance liquid chromatographic analysis , that rle contained more licochalcone a and less glycyrrhizin and isoliquiritigenin . the amount of glycyrrhizin and isoliquiritigenin was reduced by half but the amount of licochalcone a was increased to 5-fold by roasting ( data not shown ) . in this study , we first evaluated the anti - inflammatory activity of le and rle on tpa - induced ear edema to predict their antiarthritic potential . mouse ear edema induced with topically applied tpa is an excellent acute inflammation animal model , closely related with the infiltration of neutrophil and macrophages , the induction of proinflammatory cytokines including tnf- and il-1 , and the generation of ros including superoxide anion , thereby can be very useful short - term test to detect the agents with anti - arthritic potential . pretreatment with le or rle dose - dependently suppressed mouse ear edema induced with topical application of tpa for 4 hours , and rle showed more potent inhibition of inflammation than le , as shown in figure 1 . pretreatment with licochalcone a and glycyrrhizin , not isoliquiritigenin , significantly inhibited tpa - induced ear edema in mice ( data not shown ) . the increased licochalcone a by roasting may contribute to better anti - inflammatory of rle on tpa - induced acute inflammation as compared with le . we further determined the effects of le and rle on chronic inflammatory diseases using a mouse cia model . although the murine cia model does not completely recapitulate human ra , it is a useful tool for studying inflammation , autoimmune arthritis , cartilage destruction , and bone erosion . our data indicated that oral administration of le and rle to cii - immunized mice reduced the progression of arthritis by inhibiting the increase in arthritis score and paw swelling , as compared to the vehicle - treated immunized mice ( figure 2(a ) ) . there was no significant difference between the ability of le and rle to inhibit cia severity , but rle seemed to suppress early paw swelling more effectively than le . the joints of the vehicle - treated mice had noticeable pathological changes , including cia - characteristic synovial hyperplasia , infiltration of inflammatory cells into the joint cavity , and extensive pannus formation . the destruction of articular cartilage and subchondral bone was also observed in the joint tissues of vehicle - treated cia mice . the pathological events apparent in vehicle - treated cia mice were substantially reduced in the joints of le- and rle - treated cia mice ( figure 2(b ) ) . furthermore , immunohistochemical analysis indicated that the joint specimens from vehicle - treated cia mice strongly stained for mmp-3 , which is a key contributor to the destruction of cartilage , but treatment with le and rle reduced the expression of mmp-3 in inflammatory articular cartilage ( figure 2(b ) ) . similarly to human ra , the binding and accumulation of anti - cii antibodies in the articular region of cii - immunized mice may initiate inflammatory responses by activating the complement cascade . the activation of the complement cascade results in the recruitment of neutrophils and macrophages , which secrete chemotoxic materials and pro - inflammatory cytokines , such as il-1 , tnf- , il-8 , il-6 , nitric oxide , and prostaglandin e2 . of these , tnf- drives early joint inflammation and inflammatory cell infiltration into the synovial tissue , while il-1 plays a pivotal role in mediating cartilage degradation and bone erosion . , controlling mmp activity as well as the production of pro - inflammatory cytokines is required for the effective treatment of arthritis . in this study , tnf- and il-1 levels were measured in the sera of control mice and cia mice treated with vehicle , le or rle on day 45 . while tnf- and il-1 levels were significantly increased in the sera of vehicle - treated cia mice , orally administrated le and rle inhibited the cia - induced increases in the serum levels of these cytokines ( figure 3(a ) ) . these results suggest that le and rle may ameliorate cia by blocking the increased levels of tnf- and il-1 in cia mice . the prevalence of cii - specific t cells , the primary mediators of cia induction , has been observed in the early phase of ra . although the pathways that drive cytokine release in ra synovium are still undefined , t cells can regulate cytokine secretion by directly releasing il-17 into the extracellular space or via cellular contacts with synovial macrophages [ 28 , 30 ] . to estimate the effects of our extracts on cii - specific t cell responses , cell proliferation was investigated with spleen cells isolated from control mice and cia mice treated with vehicle , le or rle . the spleen is the major site of adaptive immune response to blood - borne antigens due to the high population of resident phagocytes and lymphocytes . spleen cells are stimulated with either cii to activate t cells or lps to potently activate macrophages . the proliferation of spleen cells from vehicle - treated cia mice was increased by cii stimulation for 72 hours , but spleen cells from le- and rle - treated cia mice exhibited significantly less proliferation than those from vehicle - treated cia mice ( see figure 3(b ) ) . next , cii- and lps - induced cytokine levels were measured in spleen cells from cia mice treated with vehicle , le and rle . cii- or lps - stimulated secretion of tnf- and il-1 was also reduced in spleen cells isolated from cia mice treated with le or rle , as compared to spleen cells from vehicle - treated cia mice ( figure 3(b ) ) . these results suggest that le and rle alleviate cia by reducing the immune responses of spleen cells , which results in decreased cytokine production . ros released from infiltrating inflammatory cells is implicated in the breakdown of cartilage and bone in ra , and elevated levels of mda have been observed in the serum and synovial fluid of ra patients . moreover , chronic inflammatory conditions reduce a body 's antioxidant capacity by affecting a variety of endogenous ros scavenging proteins , enzymes , and chemical compounds . therefore , control of ros levels is also required to reduce cia severity and tissue damage . to study the protective effects of le and rle on tissue oxidative damages in cia mice , mda level as a marker of lipid peroxidation , gsh content , and antioxidant enzyme activity were measured in liver and kidney tissue homogenates from control mice and cia mice treated with vehicle , le or rle . mda levels were elevated in liver and kidney tissues from vehicle - treated cia mice , but the increase in mda levels was inhibited in those from le or rle - treated cia mice ( figure 4(a ) ) . in addition , the decreased tissue gsh levels ( figure 4(b ) ) and catalase activities ( figure 4(c ) ) in vehicle - treated cia mice were rescued by oral administration of le or rle . these results suggest that le and rle can prevent tissue damage by blocking oxidative stress in cia mice . topical application of le or rle onto the mouse ear prior to tpa treatment inhibited tpa - induced acute inflammation , and oral administration of le or rle effectively suppressed the inflammatory response and tissue damage in the cia mouse model . rle exhibited a more potent inhibition on tpa - induced acute inflammation than le , but the anti - arthritic effect of rle was similar to that of le in the cia model . overall , these data suggest that supplementation with le and rle may be beneficial in preventing and treating both acute and chronic inflammatory conditions .
the anti - inflammatory activity of licorice ( le ) and roated licorice ( rle ) extracts determined in the murine phorbol ester - induced acute inflammation model and collagen - induced arthritis ( cia ) model of human rheumatoid arthritis . 
 rle possessed greater activity than le in inhibiting phorbol ester - induced ear edema . 
 oral administration of le or rle reduced clinical arthritis score , paw swelling , and histopathological changes in a murine cia . 
 le and rle decreased the levels of proinflammatory cytokines in serum and matrix metalloproteinase-3 expression in the joints . 
 cell proliferation and cytokine secretion in response to type ii collagen or lipopolysaccharide stimulation were suppressed in spleen cells from le or rle - treated cia mice . 
 furthermore , le and rle treatment prevented oxidative damages in liver and kidney tissues of cia mice . 
 taken together , le and rle have benefits in protecting against both acute inflammation and chronic inflammatory conditions including rheumatoid arthritis . 
 rle may inhibit the acute inflammation more potently than le .