ulcerative colitis ( uc ) is an idiopathic disorder that involves a proximal extension of chronic inflammation in the colonic mucosa . major manifestations of uc include bloody diarrhea , rectal bleeding , frequent stools , tenesmus , and pain in the lower abdomen , all of which undergo repeated relapses following exacerbation and remission of the disorder [ 1 , 2 ] . although the exact etiology of uc is yet to be fully understood , the general consensus is that a combination of the following factors leads to its development : genetic risks of maternal inheritance , environmental variability , and dysbiosis [ 3 , 4 ] . treatment of the disease mainly consists of conventional pharmaceutical approaches , as well as surgical interventions for those who do not respond to the initial therapeutics . for uc patients exhibit an impaired quality of life , treatment is mostly aimed at restoring it , through induction and maintenance of the remission phase . drugs currently in use for patients with uc can be categorized as anti - inflammatory drugs , immunosuppressants , pro / antibiotics , and biologic agents . however , in most cases , the underlying mechanisms are still not clear , and long - term efficacy is achieved in only approximately one - third of the patients . current options are also hindered by undesired adverse effects such as metabolic changes , increased risk of infections or lymphomas , or even higher mortality . therefore , it is imperative that effective complementary and alternative measures be developed to remedy such defects . animal models of uc have proven to be useful in terms of understanding the pathophysiology of uc , not to mention testing the in vivo potency of possible therapeutic agents . while murine models are most frequently used , a uc - like phenotype is induced via chemical administration or bacterial infection [ 4 , 7 ] . dextran sulfate sodium ( dss ) , trinitrobenzene sulfonic acid ( tnbs ) , oxazolone , and acetic acid are major colitogenic substances that cause injury to the intestinal epithelium . the most widely used is the dss , since it is relatively simple in regard to administration ( usually through drinking water ) , besides control of dosage and duration . rather than directly causing luminal inflammation per se , dss exposes the lamina propria and submucosa to enteric antigens and bacteria by breaking down the epithelial barrier . many of the consequent inflammatory responses resemble those of the human uc , as evidenced by exhibition of characteristic features such as weight loss , diarrhea , fecal occult blood , and intestinal shortening [ 9 , 10 ] . histopathologic features of dss - induced colitis include mucin depletion , epithelial degeneration , and neutrophil infiltration . the pathogenesis of dss - induced colitis involves defective alterations in the tight junction complex and marked increases in the expression of inflammatory cytokines [ 10 , 11 ] . with such keen resemblance to the human - derived uc , the preclinical model of dss - induced colitis has been validated as relevant for the translation of murine data into human disease [ 1 , 7 ] and thus has been adopted in this study . the use of complementary and alternative medicine ( cam ) in patients with inflammatory bowel disease ( ibd ) is quite common , with a rate over 40% in north america . herbal therapy is the single most used modality of cam , followed by homeopathy , naturopathy , and dietary supplements [ 13 , 14 ] . the primary reason behind such unconventional medicine use has been reported to be direct disease - related benefits , in addition to psychological factors such as the need for greater autonomy or emphasis on holistic approaches [ 14 , 15 ] . unpurified herbal decoctions contain biologically active compounds that may display antioxidative , anti - inflammatory , and prebiotic properties [ 13 , 16 ] . in this study , we have tested the effects of oral angelica acutiloba kitagawa ( aak ) extract on a dss - induced colitis model . the japanese aak belongs to the same genus as the korean a. gigas and the chinese a. sinensis , all of which have been substituted by one another for angelicae radix ( angelica root ) . while a. gigas is well - known for its active ingredient decursin and decursinol angelate , aak is differentiated by a relatively low content of both , and a relatively high content of essential oils , organic acids , and polyphenols like z - ligustilide , n - butylidenephthalide , ferulic acid , and chlorogenic acid . previously reported bioactivities of the aak extract include antitumor , anticomplementary , immunostimulatory , antidiabetic , antiobesity , and , most importantly , a considerable anti - inflammatory activity [ 17 , 2426 ] . here we demonstrate for the first time that orally administered aak extract significantly suppresses intestinal inflammation in the dss - induced acute colitis model . our findings may represent a novel efficacious and reasonable confrontation for the standard treatment of uc , as its cost of nonadherence and opportunity loss is substantial , and still on the rise . male , 6-week - old c57bl/6 mice weighing at an average of 23  g ( 1  g ) were obtained from central lab animal inc . mice were housed in a pathogen - free facility with a light / dark cycle of 12  h , under ambient temperature ( 2123c ) and humidity ( 5560% ) . all animals had access to food and water ad libitum and were acclimated for 2 weeks before the experiment . dried root of aak ( 500  g ) was obtained from kyung hee herb pharm ( seoul , korea ) . briefly , the sections were immersed in 30% etoh and extracted 2 times for 2  h each , using a reflux extractor ( glhmp - f1000 , global lab , siheung , korea ) . the extract was further concentrated with an evaporator ( rotavapor r-220 , bchi labortechnik ag , flawil , switzerland ) , followed by a filtration step and subsequent freeze - drying ( lp30 , ilshin biobase co. , yangju , korea ) at 5  mm torr ( yield = 21.3% ) . for chromatographic separation , 100  mg of freeze - dried sample powder was dissolved in 1  ml meoh , sonicated for 30  min , and filtered through a pvdf membrane . chromatographic separation of the extract was performed using a separations module ( 2690 , waters , ma , usa ) and 5  m column ( nucleosil 100 - 5 c18 , macherey - nagel , dren , germany ) . using solvents a ( water ) and b ( acetonitrile ) , the gradient program was run as follows : 03  min ( 20% b ) , 38  min ( 30% b ) , 818  min ( 30% b ) , 18 - 19  min ( 50% b ) , 1940  min ( 50% b ) , and 40 - 41  min ( 90% b ) , at 1  ml / min flow velocity and 330  nm wavelength . quantitative content of decursin ( # kp013 , npc biotech , sejong , korea ) was analyzed via a photodiode array detector ( 996 , waters , ma , usa ) interfaced to the separations module ( figure 1 ) . after adaptation , mice were randomly divided into the following 4 groups ( 46 mice / group ) : normal control , colitic control , aak 100 , and aak 500 . colitis was induced with dss as previously described . briefly , 3650  kda dss ( # 0216011080 , mp biomedicals , ca , usa ) was dissolved in sterile tap water at a final concentration of 4%  ( wt / vol ) and presented to mice as drinking water for 7 consecutive days . normal controls were administered sterile tap water by oral gavage once per day , with free access to drinking water ; colitic controls were also administered sterile tap water by oral gavage once per day , but with free access to dss - treated drinking water ; aak 100 and aak 500 mice were , respectively , administered 100 and 500  mg / kg of body wt aak by oral gavage once per day , with free access to dss - treated drinking water . the doses of aak were chosen on the evidence of previously - demonstrated protective effects of angelica root in multiple inflammatory disease models [ 22 , 2931 ] . mice were monitored daily for changes in body weight , stool consistency , and stool blood . body weight on any particular day was calculated as the relative percentage weight to that of day 1 . stool consistency was evaluated macroscopically using the following 4-point scale : 0 , formed and hard ; 1 , formed but soft ; 2 , loose stool ; 3 , watery . traces of occult blood in feces were assessed using hemoccult sensa single slides ( # 38078 , beckman coulter , ca , usa ) in the following 4-point scale : 0 , hemoccult negative (  ) ; 1 , hemoccult positive ( + ) ; 2 , hemoccult positive ( + + ) ; 3 , gross bleeding on the anus site . after sacrifice , the colon was removed and measured in length for evaluation of shortening . after 6  h receiving the last gavage , mice were anaesthetized with avertin . upon dissection , the full colon was also removed and measured in length , from the cecocolic junction to the proximal rectum . following extraction of the cecum , the colon was then washed in phosphate buffered saline ( pbs ) , and the inflamed distal region was cut and fixed in 4% formaldehyde for histological examination . the remaining mid - proximal portion was dissected into fragments and stored at 80c for further analysis . fixed distal colon tissue samples were embedded in paraffin using a tissue processor ( tp1020 , leica biosystems , nussloch , germany ) . paraffin blocks and 4  m section slides were made via conventional methods . upon hematoxylin and eosin ( h&e ) staining , representative regions of the longitudinal and transverse sections were viewed using light microscopy at 100x/400x magnification , respectively . briefly , a total score ranging from 0 ( healthy ) to 12 ( extensive injury ) was determined by the sum of respective scores for the following parameters : ( 1 ) epithelial erosion , ( 2 ) goblet cell depletion , ( 3 ) inflammatory cell infiltration , and ( 4 ) crypt deformation . scoring of each parameter was defined as follows : 0 , none ; 1 , mild ; 2 , moderate ; 3 , severe . colon tissue samples were thawed and total rna was extracted using the r&a - blue kit ( # 17501 , intron biotech . , eluted rna samples were quantified with the nanodrop spectrophotometer ( thermo scientific , de , usa ) at 260  nm absorbance , and 2  g of rna was subsequently reverse - transcribed using the primescript cdna synthesis kit ( # 6110a , takara , shiga , japan ) according to the manufacturer 's protocol . real - time quantitative pcr was carried out on reaction tubes ( # 4358293 , applied biosystems , ca , usa ) and caps ( # 4323032 , applied biosystems , ca , usa ) in a stepone real - time pcr system ( applied biosystems , ca , usa ) with 100  ng of cdna , the sensifast sybr hi - rox kit ( # bio-92005 , bioline , london , uk ) , and previously described primers for the targeted genes [ 33 , 34 ] . relative gene expression was calculated via the comparative ct ( 2 ) method , and mouse glyceraldehyde 3-phosphate - dehydrogenase ( gapdh ) was used as an endogenous control for normalization . upon whole blood collection via cardiac puncture , the serum was isolated by centrifugation at 1,500  rpm for 20  min at 4c . determination of serum ige levels was performed using the bd opteia mouse ige elisa set ( # 555248 , bd biosciences , ca , usa ) , according to the manufacturer 's protocol . after the final step , plate was read with a microplate reader ( versamax , molecular devices , ca , usa ) at 450  nm absorbance . all statistical analyses were performed using the graphpad prism 5 ( graphpad software inc . , ca , usa ) software . statistical significance for differences between curves was tested using two - way analysis of variance ( anova ) , followed by bonferroni correction . statistical significance for differences between means was tested using one - way anova , followed by newman - keuls multiple comparison test . data are presented as the mean  sd , and p values of < 0.05 , < 0.01 , and < 0.001 were considered statistically significant . upon receiving dss , mice developed acute colitis , which was evidenced by a reduction in average food intake and body weight . oral administration of aak ( 500  mg / kg / day ) resulted in a higher daily average food intake compared to colitic controls with vehicle treatment ( figure 2(a ) ) . accordingly , significant reduction in body weight loss was observed in mice with aak ( 500  mg / kg / day ) administration , on days 5 , 6 , and 7 ( figure 2(b ) ) . no significant differences between low - dose ( 100  mg / kg / day ) aak - treated mice and colitic controls were observed , in both average food intake and body weight . changes in stool consistency and occult blood were observed in mice upon exposure to dss . as early as day 2 , mice receiving dss developed soft stools with mild traces of diarrhea and tested positive in the guaiac test . the severity of manifestations progressively intensified towards termination of experiment , where mice exhibited watery feces and gross bleeding on the anus site . in line with the effects on food intake and body weight loss , oral administration of aak ( 500  mg / kg / day ) resulted in a significant suppression of diarrhea events on days 3 , 6 , and 7 ( figure 2(c ) ) . oral aak ( 500  mg / kg / day ) also significantly protected against presence of occult blood on days 6 and 7 ( figure 2(d ) ) . again , no significant differences between low - dose ( 100  mg / kg / day ) aak - treated mice and colitic controls were observed , in the scores of both stool consistency and occult blood . immediately after the termination of experiment , colitic controls with dss had a significantly shortened colon length ( 5.1  0.4  cm ) , compared to normal controls with vehicle treatment ( 7.3  0.5  cm ) . mice with oral aak ( 500  mg / kg / day ) administration had a significantly well - retained colon length ( 6.6  0.3  cm ) compared to colitic controls . as for the low - dose ( 100  mg / kg / day ) aak group , average colon length ( 5.1  0.6  cm ) was analogous to that of colitic controls ( figures 3(a ) and 3(b ) ) . in the case of spleen weight , oral aak ( 500  mg / kg / day ) administration showed a trend of reduction ( 68.9  3.8  mg ) , although it did not reach statistical significance compared to colitic controls ( 84.1  3.3  mg ) ( figure 3(c ) ) . histological assessment of distal colon samples revealed that dss - treated mice exhibited loss of surface epithelium , villous blunting , crypt distortion or loss , goblet cell loss , and inflammatory cell infiltration . while normal controls showed mild or no signs of histological damage ( score 0.5  0.6 ) , colitic controls had marked severe morphological changes throughout the epithelium and mucosa , with a dense transmural inflammatory cell infiltration ( score 10.5  2.4 ) . the colons of oral aak - treated mice showed substantial protection against such damage . at high - dose ( 500  mg / kg / day ) aak administration , mucosal architectures and goblet cells were relatively well - preserved , with a sparse distribution of inflammatory cells in the lamina propria ( score 4.7  1.9 ) . low - dose ( 100  mg / kg / day ) administration of aak did exhibit signs of amelioration , but not to the same extent ( score 8  0.8 ) ( figure 4 ) . to evaluate the mitigative effects of aak on dss - mediated immune response , expression of inflammatory cytokines , inducible enzymes , matrix metalloproteinases ( mmps ) , and tight junction - related proteins ifn- , il-1 , il-6 , il-12 , and il-10 , compared to normal controls . administration of aak ( 500  mg / kg / day ) was able to significantly suppress the elevation of all these cytokines ( figure 5 ) . regarding the expression of inducible enzymes inos and cox-2 , oral aak ( 500  mg / kg / day ) significantly suppressed both , which were elevated upon dss administration . similar patterns of amelioration were observed in the expression of mmp-9 and mmp-14 ( figure 6 ) . while degraded epithelial integrity was evident in the marked decrease of occludin and zo-1 expression upon dss administration , oral aak ( 500  mg / kg / day ) was capable of restoring it . in the case of icam-1 , an increase was present upon dss administration , which was successfully reduced to the basal level in oral aak ( 500  mg / kg / day)-treated mice ( figure 7 ) . in dss - treated colitic controls , serum total ige levels were upregulated by approximately twofold ( 484.4  44.4 ng / ml ) , compared to that of normal controls ( 168.6  60.2  ng / ml ) . in mice with oral aak ( 500  mg / kg / day ) administration , serum total ige levels were significantly downregulated ( 224  172.3  ng / ml ) ( figure 8) . clinical response to standard therapy among uc patients is discrete , depending on the combination of factors that eventually led to the disease . despite recent advances in therapeutic regimen , a considerable portion of patients with uc still fail or lose response to conventional nonbiologic agents , which results in varied complications such as fibrostenosis that may require surgical intervention . approximately 11% of patients who use immunomodulators such as azathioprine or methotrexate discontinue administration due to allergic reactions , nausea , or pancreatitis . in the case of corticosteroids , 55% of patients end up in cessation of therapy as a result of recurring acne , facial swelling , and osteoporosis . while biological agents ( e.g. , anti - tnf monoclonal antibodies ) emerge as an alternative option , they have also been held by secondary responses that include the risk of hypersensitivity , immunogenicity , infection , and congestive heart failure . blockade of a single cytokine as such may give rise to an alternative compensatory pathway and thus is ineffective in many cases . herbal decoctions comprise a number of components that modulate the expression of multiple cytokines simultaneously   and therefore could prove as an effective complementary or alternative modality for uc patients . nonadherence to treatment is yet another problem in the standard management of uc , which is caused by inconveniences following administration of large number of tablets , fear of side effects , or doubts in the need for medication during disease quiescence . as phytotherapy is highly accepted by uc patients for its efficacy , low cost , and relative safety , it may also prove as a promising confrontation for such patient - derived nonadherence issues . in this study , we demonstrated the effects of aak extract in a dss - induced acute colitis model . roots of the angelica plant are known to be rich in polysaccharides that exert immunomodulatory   and anti - inflammatory [ 24 , 25 ] effects . the polysaccharides from a. sinensis , which is largely similar in composition to that of aak , showed protective effects in tnbs and dnbs - induced rat models of uc [ 29 , 40 , 41 ] . while the root extracts from aak have been reported to suppress inflammatory mediators in mouse macrophages , analogous effects on a murine uc model are not yet confirmed . hence this study was aimed at investigating the effects of aak extract on a dss - induced model of uc . as previously reported , intestinal inflammation in the dss - induced model of uc is primarily manifested as symptoms comparable to those of human - derived uc , such as anorexia , body weight loss , diarrhea , fecal occult blood , and colon shortening [ 9 , 10 , 42 ] . in patients with ibd , decreased appetite in uc patients has been reported to be the result of increased serum leptin levels , which in turn sends a negative feedback signal to the hypothalamus [ 44 , 45 ] . as for animal models , a comparable elevation in serum leptin , as well as the consequent loss of body weight , has been observed in the early stages of tnbs - induced intestinal inflammation in rats . in line with these reports , the colitic group in our study exhibited a decrease in average food intake and body weight over the days of dss administration . the ethanol extract of aak was able to attenuate anorexia and subsequent weight loss , as evidenced by a relatively well - maintained food consumption and body weight at the terminal stage of experiment . events of diarrhea and fecal occult blood were also significantly suppressed during the same period , both of which represent impaired epithelial barrier function and mucosal damage [ 47 , 48 ] . for unknown reasons , the colonic longitudinal muscle undergoes hypertrophy and subsequent contraction during chronic uc . this contraction produces a diffuse or segmental narrowing of the lumen , which in turn causes shortening of the colon , namely , the  lead pipe sign  . in this study , colonic length was significantly retained upon oral administration of aak , compared to colitic controls . in animal models of uc , splenomegaly is validly indicative of intestinal inflammation , and mitigative effects of therapeutic substances have often been confirmed by the suppression of spleen enlargement [ 50 , 51 ] . the imbalance between pro- and anti - inflammatory cytokines disrupts ease of inflammation and further leads to an elongation of the disease . in line with such reports , cytokine - based therapies such as anti - tnf- factors have recently been adopted in the uc - treatment algorithm . the murine dss - induced colitis model exhibits a distinct cytokine profile with elevated levels of tnf- , ifn- , il-1 , il-6 , and il-12 . while the models used in this study displayed similar patterns , our data suggest that oral administration of aak significantly suppresses mrna expression of such inflammatory cytokines in the inflamed colonic tissue . the proinflammatory cytokines tnf- and ifn- play a pivotal role in the pathogenesis of uc , by altering the tight junction and inducing apoptosis of intestinal epithelial cells [ 5355 ] . in caco-2 cell monolayers , tnf--induced epithelial permeability was mediated by the expression of myosin - light chain kinase ( mlck ) . in the apical membrane of t84 cells , ifn- induced redistribution of occludin , claudin-1 and claudin-4 , away from the tight junction domain . the therapeutic potency of targeting these cytokines have been demonstrated in actual clinical trials with anti - tnf- antibodies , as well as in in vivo studies where colitis showed attenuation upon dss stimulation in ifn- mice . in the inflamed mucosa of ibd patients , key antigen - presenting cells ( apcs ) produce considerable amounts of proinflammatory cytokines such as il-1 , il-6 , and il-12 . when compared to healthy subjects , patients with uc manifested a significant reduction in the ratio of il-1 receptor antagonist to il-1 , which implies the augmented activation of il-1 system in uc . in a previous study using dss - induced model of colitis , mice deficient in il-1-converting enzyme ( caspase-1 ) exhibited attenuated signs of intestinal inflammation , suggesting the therapeutic relevance of il-1 family blockade . in experimental colitis , macrophages and cd4 t cells of the lamina propria produce an increased amount of il-6 [ 62 , 63 ] . upon binding with soluble il-6r ( sil-6r ) , the il-6-sil-6r complex induces proinflammatory cytokine production in intestinal apcs and t cells , by binding to the gp130 surface molecule [ 36 , 62 ] . the therapeutic potentials of il-6 blockade have been confirmed in a previous study by yamamoto et al . , where administration of anti - il-6r monoclonal antibody to a murine model of colitis resulted in reduced disease activity and suppression of proinflammatory cytokines . il-12 is a heterodimeric cytokine produced by monocytes and macrophages in response to bacteria or their products [ 65 , 66 ] . by inducing ifn- synthesis and th1 cell differentiation , il-12 contributes to the breakdown of tolerance against luminal antigens in human and murine colitis . since ifn- upregulates il-12 production in macrophages , the established loop between il-12 and ifn- further contributes to the perpetuation of intestinal inflammation . as neutralizing antibodies to il-12 have shown suppressive effects on chronic intestinal inflammation , targeting il-12 in uc may also prove as an effective means of treatment [ 69 , 70 ] . the anti - inflammatory cytokine il-10 is known to exhibit immunoregulatory properties by blocking the production of proinflammatory cytokines in monocytes and macrophages . restoration of endogenous il-10 , as well as administration of its exogenous components , has been proposed as a possible treatment for ibd . however , such is not the case in this study , as the level of il-10 expression was upregulated upon dss administration and downregulated by aak treatment . similar patterns have been reported in previous studies with murine [ 34 , 73 ] and porcine   models of intestinal inflammation . one possible explanation may be the rapid return to a homeostatic cytokine milieu upon treatment , as the endogenous production of il-10 may have been insufficient to downregulate proinflammatory cytokines , and thus local inflammation [ 71 , 74 ] . in further studies , the expression of anti - inflammatory cytokines besides il-10 , such as il-4 , should be examined to confirm the contribution of other endogenous factors . taken together , the suppressive effects of aak on inflammatory mediators of the above imply its possibility as a potent complementary and alternative therapy against uc . oral administration of aak also significantly suppressed the mrna expression of inducible enzymes and mmps in the inflamed tissues of dss - induced colitic mice . elevated expressions of inos and cox-2 in the morbid colonic mucosa indicate production of nitric oxide ( no ) and prostaglandin e2 ( pge2 ) , two of the bioactive agents involved in colonic injury . recent studies have suggested a synergetic action of both enzymes , as inhibition of no synthesis resulted in a reduced secretion of pge2 . combined blockage of inos and cox-2 reportedly had a cytoprotective effect under uc conditions , and analogous data were drawn in the present study using aak . mmps are endopeptidases that degrade the extracellular matrix ( ecm ) components during active flares of uc . mmps are capable of shedding molecules such as tnf- , il-1 , and il-6 from macrophage surfaces . in this study , mucosal recovery of aak - treated mice was evident in our histological data , which was also confirmed by the restored mrna expression of tight junction proteins zo-1 and occludin . lastly , our study demonstrated the suppressive effects of aak on total serum ige concentrations . patients with uc have reported suffering from allergic diseases such as asthma , allergic rhinitis , or eczema , twice as much as matched healthy controls . in another study by d'arienzo et al . , total serum ige levels were accordingly higher in uc patients , compared to healthy controls . our results indicate that oral aak extract may also have beneficial effects in treating the secondary atopic manifestations following uc . taken together , this study suggests that oral administration of aak exerts a protective effect against the pathogenesis of acute colitis , by improving major symptoms such as anorexia , weight loss , diarrhea , occult blood , and colon shortening . furthermore , we demonstrated that such attenuative effects are partially mediated by the suppression of multiple inflammatory markers , including cytokines , inducible enzymes , mmps , and tight junction - related proteins . these findings provide the possibilities for a more sustainable approach towards uc , as conventional therapeutics with a single - cytokine target are likely to have limitations due to development of compensatory pathological pathways . in the present study , aak mostly regulated apc - derived cytokines . however , recent studies have suggested that nonimmune cells , such as epithelial cells or stromal fibroblasts , may also produce inflammatory cytokines in ibd . in view of such findings , we may hypothesize that aak elicits an innate immune cell - mediated response , while targeting the epithelium itself at the same time . however , as our data do not confirm a predominant activation of either mechanism , further in vivo and in vitro experiments are required to define the exact mode - of - action responsible for the protective activities of aak .
we examined the protective effects of angelica acutiloba kitagawa ( aak ) extract on a murine model of acute experimental colitis . 
 colitis was induced by 4% dextran sulfate sodium ( dss ) in the drinking water of male c57bl/6 mice , for 7 consecutive days . 
 oral administration of aak extract ( 500  mg / kg / day ) significantly alleviated dss - induced symptoms such as anorexia , weight loss , events of diarrhea or bloody stools , and colon shortening . 
 histological damage was also ameliorated , as evidenced by the architectural preservation and suppression of inflammatory cell infiltration in colonic samples . 
 treatment improved the colonic mrna expression of different inflammatory markers : cytokines , inducible enzymes , matrix metalloproteinases , and tight junction - related proteins . in the isolated serum 
 , ige levels were downregulated . 
 collectively , these findings indicate the therapeutic potentials of aak as an effective complementary or alternative modality for the treatment of ulcerative colitis .