it was reported that the colon cancer mortalities are higher in the countries where people consume more animal fat . on the other hand ,
fiber - rich foods increase the volume of feces and promote the creeping motion of the large intestine , hence , reduce the contacting time of carcinogens in the body .
immune response factors such as cytokines , reactive oxygen species ( ros ) , proinflammatory enzyme cyclooxygenase-2 ( cox-2 ) , and nitric oxide synthase ( inos ) may accelerate the development of cancer .
it has been indicated that colon cancer progression could be slowed down by reducing the expression of cox-2 protein or reducing inflammatory reaction [ 4 , 5 ] . at the location of inflammation
, inos may cause the overproduction of nitric oxide , which results in damages on dna repair and promotes the proliferation of cancer cells . in the early stage of colon cancer in human , lowering the expression of inos could be used to reduce the formation of cancer cells .
in addition , carcinogens may increase the activities of cox-2 and inos on the mucosa of the colon , which also causes the promotion of cancer occurrence .
it was indicated that by reducing nf-b expression , the occurrence of cancer can be reduced .
there are several enzymes other than immune response factors , such as nitroreductase , azoreductase , and -glucuronidase produced by the bacteria in the intestine , that are closely related to cancer occurrence .
hence , by inhibiting the activities of those enzymes , the chance of cancer formation can also be reduced .  
soy saponins are amphiphilic compounds and can binnd to the phospholipids and cholesterol with the hydroxyl groups on the aglycone moiety of the compounds , hence , change the structure of the cell membrane .
soy saponins have been found to have inhibitory effects in vitro on cancer cells   by changing the cell morphology , cell proliferation enzymes , and cell growth . in this study
, we investigated the effect and mechanism of soy saponins on preventing colon cancer in rats by observing the formation of aberrant crypt foci ( acf ) as well as measuring the inflammatory factors , nf-b , cox-2 , and inos , and enzyme activities in the large intestine of rats .
  fifty male fisher 344 rats , six weeks old , were housed in iron cages ( 2/cage ) and kept under the 12/12 hours light - dark conditions of 50% humidity and a temperature of 23c .
ain93 g was used as the control ( basal ) diet . from week 0 , rats in ds0 , ds0.5 , and ds1 consumed the diet containing 0% , 0.5% , 1% soy saponin , respectively , for four weeks before receiving dimethylhydrazine ( dmh ) injections ( 20  mg / kg - bw , 2 times / week ) for the following 6 weeks .
rats in ps0 and ps0.5 groups consumed the basal diet plus 0% and 0.5% of soy saponins , respectively , before receiving saline injections ( 2  ml / kg  bw , 2 times / week ) for the following 6 weeks . the blood samples were collected in week 0 and week 15 . at the end of week 15 , all rats were sacrificed .  
the purity of crude saponins extracted was examined by hplc ( tsp , germany ) with commercial soy saponins as the standards ( wako , japan ) .
the hplc condition was as follows : c18 column ( vercopak , ods-3 , 4.6  250  mm ) ; uv absorbance : 190350  nm ; analyzing temperature : 30c ; flow rate : 1  ml / min ; gradient solvent system : solvent a , 0.05% trifluoroacetic acid in water and solvent b , acetonitrile .
the gradient program was set as 63% a to 52% a in 38  min .
blood samples were drown from the abdominal cavity vein and collected in tubes containing heparin at the end of the experiment .
the liver was purged with phosphate buffer saline ( pbs ) and stored in formaldehyde , followed by an observation using h & e stain .
the rectum and colon samples were collected and washed with pbs and then stored at 80c .
the average length of the rectum ( n = 4 ) of each group was recorded .
the colon was divided into 3 sections , near cecum section , middle section , and near anus section .
isoflavone standards were prepared using commercially available 25  ppm daidzin ( 30408 , fluka ) , daidzein ( wako ) , genistein ( g6649 , sigma ) and genistin ( g0897 , sigma ) .
samples , dissolved in 20  ml 80% methanol ( ms1992 , tedia ) , were prepared from 0.5  g crude soy saponins extract .
a high performance liquid chromatography ( hplc ) method was used for analysis with c18 column ( inertsil 6 , ods-3 , 4.6  250  mm , vercopak ) .
the injection volume was 20  l . the column temperature was kept constant at 40c ( super co-150 , enshine ) and the flow rate was 1  ml / min .
the binary gradient elution system consisted of two solvents : acetonitrile ( a ) and acetic acid ( 0.1% in water , b ) .
separation was achieved using the following gradient : solvent a , 14  min , 15%  35% ; 0448  min , 35% .
the isoflavones in the plasma were extracted with tert - butyl methyl ester ( tbme ) and analyzed with hplc with c18 column ( inertsil 6 , ods-3 , 4.6  250  mm , vercopak ) at 40c . the uv detection wavelength was set at 248  nm and the mobile phase was methanol  :  ammonium acetate ( 0.1  m)(40 : 60(ph  4.7 ) ) .
the plasma lipid analysis was performed by using automatic biochemical analyzer ( dri - chem 3500 , fuji ) for total triglyceride ( tg ) and total cholesterol ( tc ) determination .
a colon sample was fixed in 10% formalin for 24  hr and flushed with saline on a filter , then dipped in 0.5% methylene blue for 1  min , followed by being washed with distilled water briefly and placed on a microscope slide with the mucosal surface up .
the acfs were classified into : ( a ) normal crypt , ( b ) 1 aberrant crypt ( ac)/acf , ( c ) 2ac / acf and ( d ) 3ac / acf .
then the acfs containing 13 acs were defined as small acfs , and those containing more than 3acs were large acfs .
cell protein was extracted and determined using the commercial protein assay kit ( bca1 and b9643 , sigma ) .
briefly , 200  l of bca assay reagent ( bicinchoninic acid solution : copper sulfate pentahydrate 4% solution ( 50  :  1 ) ) was completely mixed with a sample and incubated for 30  min at 37c .
was measured at 562  nm . the cox-2 ( mw = 70  kda ) and the inos ( mw = 130  kda ) protein expressions were measured by using sds - page with 10% resolving gel and 7% stacking gel followed by western blotting while -actin ( 42  kda ) was used as an internal control .
-actin ( mouse anti--actin monoclonal antibody , a 5441 , sigma ) ; cox-2 ( goat anti - cox monoclonal antibody , sc-19999 , santa cruz ) ; inos ( rabbit anti - nos2 polyclonal antibody , sc-649 , santa cruz ) diluted in tbst ( 1  :  1000 ) which obtained 1% bsa . 
 second antibodies .
-actin ( rabbit anti - mouse igg , a9044 , sigma ) ; cox-2 ( donkey anti - mouse igg , sc-2020 , santa cruz ) ; inos ( goat anti - rabbit igg , sc-2004 , santa cruz ) .
a colon mucosa sample was homogenized and adjusted to ph 7.2 and then mixed with 24 times volume of acetone and incubated for 5  min in room temperature .
the acetone in the supernatant was removed and the pge2 was determined with commercial kit ( prostaglandin e2 eia kit , cayman ) .
the homogenization buffer was prepared by dissolving 13.69  g phosphate in ddh2o containing 357.8  mg edta and 3.6  mg indomethacin and adding ddh2o to 1000  ml ( adjust ph to 7.4 ) .
a sample of 20  l and 200  l gus ( 31.5  mg pnpg , n-1627 , sigma , dissolved in 100  ml 50  mm na3po4 , ph 7.0 ) were mixed well , followed by performing an enzyme - linked immunosorbent assay ( elisa ) at 415  nm every 10  min for an hour .
liver samples were stained with hematoxylin and eosin ( h & e ) dye and subjected to pathological observation .
data were analyzed with one - way analysis of variance ( anova ) followed by fisher 's least significant differences ( lsd ) and dunnett post hoc analysis .
one gram of the soy extract contains saponin 778.3  3.8  mg and isoflavone 2.8  0.3  mg , including daidzin 1180  44  g , genistin 923  11  g , daidzein 173  58  g , and genistein 474  197  g .
the average body weight of rats in all groups increased as the experiment proceeded but was not significant between groups .
the average change per day in body weight was 1.77  0.04  g in ds0 , 1.68  0.06  g in ds0.5 , 1.79  0.06  g in ds1 , 1.86  0.06  g in ps0 , and 1.85  0.08  g in ps0.5 .
those receiving dmh injection tended to have a lower body weight increase compared to those receiving saline injection .
the average daily food intake of rats was 16.44  0.26  g in ds0 , 15.45  0.35  g in ds0.5 , 15.95  0.33  g in ds1 , 16.78  0.24  g in ps0 , and 15.85  0.38  g in ps0.5 .
the addition of soy extract and injection of dmh did not alter the food consumption ( figure 1 ) .
the percentage of the liver weight in body weight was 3.80  0.09% in ds0 , 3.77  0.15% in ds0.5 , 3.41  0.13% in ds1 , 3.79  0.12% in ps0 , and 3.58  0.19% in ps0.5 , and no statistical difference was found .
no significant difference in the length and weight of the colon was detected among groups either .
small  acfs , and those with more than 3  ac are  large  acfs .
our results show that the numbers of small acfs were negatively correlated to saponin dosages ( p < 0.05 , table 2 ) . also , rats in ds1 group had a lower count of large acfs ( > 3 acs ) than those in ds0 group did ( p < 0.05 , table 3 ) .
the results also show that there were aberrant crypt foci ( acfs ) with 16 aberrant crypts ( ac ) in the ds0 , ds0.5 , and ds1 groups ( figure 2 ) .
no acfs were found in the ps0 and ps0.5 groups .   on the other hand
, the ds1 group had a lower number of total acfs in the cecum region than the ds0 group did ( p < 0.05 ) . in the middle section of the colon ,
ds1 group had less acfs than ds0 group and ds0.5 group ( p < 0.05 ) .
at the anal end , the ds0.5 group and the ds1 group had lower total acfs than the ds0 group ( p < 0.05 ) . in the whole colon section
, the acfs numbers were negatively correlated to the dose of saponin treated in the experiment ( p < 0.05 ) ( table 4 ) .   
after the injection of dmh , the expressions of cox-2 protein were more significant in the saponin - treated groups ( ds0 , ds0.5 , and ds1 ) than the ps groups , however , not significant among the ds0 , ds0.5 , and ds1 groups , and there was no cox-2 expression in the ps groups .
there was no significant difference in the colon section regarding the expression of cox-2 , while no change in inos expression was detected in the experiment ( figures 3(b ) and 3(c ) ) .
the level of pge2 in ds0 was significantly higher than that in ps0 ( p < 0.05 ) .
there was no significant difference among ds0 , ds0.5 , and ds1 ( figure 4 ) .  
the relative activity of -glucuronidase of the colon in ds0 was 346.9  15.6  nmole / min / mg protein , significantly higher than those in ps0 ( 158.4  8.8  nmole / min / mg protein ) , ds0.5 ( 209.6  17.1  nmole / min / mg protein ) , and ds1 ( 163.7  5.2  nmole / min / mg protein ) ( p < 0.05 ) ( figure 5 ) .
soy compositions have been shown to have anticancer effects , and the saponins are ones of those that are effective . in this experiment , we demonstrated the effect of the soy saponin consumption on preventing the formation of colon cancer .
it was indicated that the size and number of aberrant crypt foci ( acfs ) increased as the experiment time increased .
also , the number of acfs can be an indicator for acfs development . in this experiment , after the injection of dmh , the number of total acfs was 27.0  1.9 , in which the largest one had 6 ac .
thus , with the results of acfs development , it was applicable to evaluate the effect of saponins on the prevention of colon cancer .
the number of acfs on the colon mucosa is affected by the type , age , and sex of the animal , frequency and dosage of the carcinogen injected , the sections in the colon , and the personnel who is involved in counting acfs .
the mostly used carcinogen for inducing colon cancer in the animal model is azoxymethane ( aom ) , the metabolite of dimethylhydrazine ( dmh ) but more toxic than dmh .
it was found that the injection of 20  mg / kg  wt of dmh for 5 weeks resulted in 2830 acfs [ 18 , 19 ] .
the injection of aom in similar dosages produced much more severe symptoms , in which more than 200  acfs were induced [ 20 , 21 ] . in our experiment , rats received 20  mg / kg  wt of dmh twice a week for 6 consecutive weeks that resulted in an average of 27  acfs / rat with 16 acs induced .
thus , using dmh was successful in inducing acfs and can be used as an alternative method for colon cancer research when using an animal model .
in addition to acfs , the biomarkers for determining the early stages of colon cancer may include mucin - depleted foci ( mdf ) , beta - catenin - accumulated crypts ( bcac ) , and dysplastic acf ( dacf ) [ 19 , 22 , 23 ] .
however , acfs are now used frequently as markers of cancer due to the simplicity in performing without tissue biopsy and the results usually being closely related to the occurrence of tumor .
it was shown that soy saponin was effective in inhibiting the growth of the ht-29 human colon cancer cell line by changing the cell morphology and the cell proliferation enzymes .
it was indicated that the numbers of total and large aberrant crypt foci ( acf ) are reliable indicators of colon cancer occurrence .
we found that as the dosage of soy saponin extract in the diet increased , the number of total acfs decreased .
soy saponins were also found to have cancer - protecting ability by reducing the expression of mrna and the amount of matrix metalloproteinases ( mmp-2 and mmp-9 ) in ht-1080 cells .
it was also shown that triterpenoid b - group soy saponins may induce the autophagic capacity of htc-15 cells by modulating the activity of extracellular regulated kinase 1/2 ( erk1/2 ) .
however , it was also indicated that the formation of acfs was more related to a suppressed apoptosis of cancer cells by way of modulating the epidermal growth factor ( egf ) signaling pathway rather than the phosphorylated akt and erk .  
soy isoflavones , different from saponins that can not be absorbed by the small intestine , also have been found to reduce the occurrences of several cancers including prostate cancers and breast cancers [ 29 , 30 ] . a dose of 40  mg / kg  bw successfully decreased the tumor burden and tumor size in sd rats . in our experiment
, it was due to the fasting time ( 12  hours ) and the short half - life of isoflavone in rats ( 8.4  hr for genistein and 5.8  hr for daidzein ) before the blood was drawn at each time period , that no isoflavone was detected in the blood samples .
also , 1 gram of the soybean saponin extract contained 778.3  mg of saponin and 2.75  mg of isoflavone , including daidzin 1180  44  g , genistin 923  11  g , daidzein 173  58  g , and genistein 474  197  g .
rats in the group of 1x soy saponin extract ( ds1 ) consumed 0.16  g soy saponin and 0.55  mg isoflavone ( daidzin 0.24  mg , genistin 0.17  mg , daidzein 0.03  mg , and genistein 0.09 
it was found that 6,7,4-trihydroxyisoflavone ( 6,7,4-thif ) , a metabolite of daidzein , inhibited the growth of hct-116 human colon cancer cells .
6,7,4-thif also arrested the cell cycle of hct-116 at the s and g2/m phases and reduced the expression of cyclin - dependent kinase 2 ( cdk2 ) .
isoflavone may also inhibit the growth of colon cancer cells , dld-1 , by increasing the expression of estrogen receptor ( er)- . although the isoflavone in the diet might not be enough to lower the risk of colon cancer , but together with saponins
it was indicated   that the total blood cholesterol and triglyceride in c57b1/6j mice were decreased by consuming the high - fat diet containing 0.2% of isoflavone a day .
the structure of soy saponins can bind to cholesterol and reduce the absorption of both exogenous and endogenous cholesterol as well as the resorption of bile acid from the gut .
however , in our experiment , since the diets were not high - fat diets and the blood lipid level in each group was still in a normal range throughout the experiment period , there was no significant difference in the changes of blood triglyceride and total cholesterol among each group ( table 5 ) .
cyclooxygenase ( cox ) and inducible nitric oxides ( inos ) have been used as the index of colon cancer occurrence . among three types of cox ( cox-1-cox-3 )
only cox-2 gene expression was found to increase in human colon cancer . during a cancer progression stage , cox-2 activity significantly elevates .
inhibition of cox-2 activity may be a preventive method of colon cancer . in this investigation , however , cox-2 and inos activities did not change significantly , which may be due to a not - long - enough experiment period or a not sufficient amount of protein produced . in recent years
, -glucuronidase activity in the colon has become one of the major indices in the etiology of colon cancer .
it was shown that certain types of dietary fiber may have colon cancer preventive effect via changing the secondary bile acids resorption and reducing the activity of bacterial enzymes such as -glucuronidase , which plays a role in carcinogenesis .
rats fed with high - fat diet also resulted in elevated levels of bacterial -glucuronidase activity in the large intestine .
it has been found that -glucuronidase activity may be the most important key factor in the genotoxicity of the food - caused colon cancer . in our experiment ,
the activity of -glucuronidase was significantly increased after the injection of dmh and was reduced by consuming the diet containing soy saponins , which indicates that soy saponins can be one of the ingredients for colon cancer prevention . in conclusion , we found that soy saponins reduced the number of acfs by reducing the activity of -glucuronidase in colonic mucosa but not the cox-2 protein expression and pge2 level .
 
 objective . the effect of extracted crude soybean saponins on preneoplastic lesions , aberrant crypt foci ( acf ) , and the related mechanism were investigated . 
 
 research methods and procedures . 
 rats were assigned into five groups according to different doses of extracted crude soybean saponins and received 1,2-dimethylhydrazine ( dmh ) injection in week 5 . in week 15 , all rats were sacrificed . 
 the number of acfs , the cyclooxygenase-2 ( cox-2 ) protein expression , the level of prostaglandins e2 ( pge2 ) , and the activity of -glucuronidase were examined . 
 results . 
 results revealed that the consumption of extracted crude soybean saponins decreased the number of acfs and the activity of -glucuronidase in rats , while the expression of cox-2 protein and pge2 level were not affected . 
 
 conclusions . 
 soybean saponins were effective in inhibiting colon cancer by downregulating the activity of -glucuronidase in colonic mucosa but not the cox-2 protein expression and pge2 level . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion