lectins are carbohydrate - binding proteins , which are highly variable in their amino acid sequences , widely distributed in microorganisms , viruses , animals and higher plants , and with different functions , structures , tissue localizations and carbohydrate - binding specificities .
plant lectins were classified into seven families according to their evolutionary and structural characteristics by van damme et al .
the numerous lectins studied , those of the legumes occupy a special position , since they represent the largest and most thoroughly studied family of these proteins .
the leguminous seed lectins show a remarkable conservation in primary , secondary , and tertiary structures . because of the very similar tertiary structures and varied quaternary structures , legume lectins have become an excellent model to analyze the role of primary structure in determining the modes of quaternary association .
a number of diverse physiological roles have been proposed for these proteins , including mitogenic [ 5 , 6 ] , antifungal   , antiviral [ 8 , 9 ] , anti - insect [ 10 , 11 ] , and antitumor [ 6 , 1215 ] activities .
their sugar - binding properties are also used as molecular tools to study glycoconjugates or for drug delivery and targeting [ 16 , 17 ] .  
there are many problems to overcome during preparation of lectins , such as a low extraction rate , purification difficulties , and high extraction cost , which restricts the application of lectins .  
lectin extraction is usually achieved using different methods of diffusion in aqueous solution and ammonium sulfate precipitation .
problems with these methods include cumbersome operations , a low extraction rate , hard to extract hand - running and amplify , which universally exist in the separation and purification of biomolecules .
to solve these problems , alternative techniques have received particular attention , such as liquid - liquid extraction with reversed micelles . in this paper ,
reversed micelles were used and compared with traditional methods for the extraction of red kidney bean lectin .  
polysaccharides isolated from a variety of traditional medicinal plants can greatly stimulate immune system in vivo and in vitro , therefore , they have the potential to be immunomodulators with wide applications [ 1820 ] .
most polysaccharides derived from traditional medicinal plants are relatively nontoxic and do not cause significant side effects , which are major problems associated with immunomodulatory bacterial polysaccharides and synthetic compounds .
thus , plant polysaccharides are ideal candidates for therapeutics   and three polysaccharides ( lentinan , schizophyllan and krestin ) were accepted as immunoceuticals in several oriental countries in 2002 . in this work
, mitogenic activity of red kidney bean lectin and four chinese herbal polysaccharides ( chps ) were applied together to detect if the immunomodulatory activities were enhanced .
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) and electrophoretic reagents were purchased from sigma ( sigma aldrich china ) .
six- to eight - week - old female kunming mice , each weighing approximately 20  g , were obtained from the laboratory animal center in the 2nd affiliated hospital of harbin medicine university .
( 1 ) soaking methodred kidney beans were ground to a powder in an electric mill and filtered through 80 mesh grit . the powder ( 5  g )
was mixed with 0.15  m nacl ( 1:8 , w / v ) for 48 h at   4c , and filtered through 80 mesh grid . subsequently , the filtrate was centrifuged at 9168  g for 30 minutes , and the supernatant was fractionally precipitated with ammonium sulfate at 40% , 50% , 60% , and 70% saturation , respectively .
the four pellets were combined , dissolved in a minimal volume of water , and dialyzed against distilled water at   4c . 
 
red kidney beans were ground to a powder in an electric mill and filtered through 80 mesh grit .
the powder ( 5  g ) was mixed with 0.15  m nacl ( 1:8 , w / v ) for 48 h at   4c , and filtered through 80 mesh grid .
subsequently , the filtrate was centrifuged at 9168  g for 30 minutes , and the supernatant was fractionally precipitated with ammonium sulfate at 40% , 50% , 60% , and 70% saturation , respectively .
the four pellets were combined , dissolved in a minimal volume of water , and dialyzed against distilled water at   4c .  

( 2 ) degreasing methodthe powder was defatted twice with sherwood oil ( 1 : 8 , w / v ) for 4 h at 4c , which was removed by volatilization at room temperature , to obtain degreased bean powder .
the degreased bean powder was mixed with 0.15  m nacl ( 1:8 , w / v ) for 48 h at 4 c , and subsequent procedures were identical to those described in section 2.2.1.(1 ) . 
 
the powder was defatted twice with sherwood oil ( 1 : 8 , w / v ) for 4 h at 4c , which was removed by volatilization at room temperature , to obtain degreased bean powder .
the degreased bean powder was mixed with 0.15  m nacl ( 1:8 , w / v ) for 48 h at 4 c , and subsequent procedures were identical to those described in section 2.2.1.(1 ) .  

( 3 ) homogenate methodred kidney beans ( 5  g ) were softened in 400  ml nacl ( 1:8 , w / v ) for 24 hours at 4c , then homogenized in a blender .
the homogenate was diffused for a further 24 hours at 4c , then filtered through an 80 mesh grid .
the filtrate was centrifuged at 9168   g for 30 minutes , and the supernatant was precipitated hierarchically by ammonium sulfate.to evaluate the protein concentration and hemagglutinating activity of each extract , all extracts were adjusted to the same volume using polyethylene glycol 20000 when necessary . 
 
red kidney beans ( 5  g ) were softened in 400  ml nacl ( 1:8 , w / v ) for 24 hours at 4c , then homogenized in a blender .
the homogenate was diffused for a further 24 hours at 4c , then filtered through an 80 mesh grid .
the filtrate was centrifuged at 9168   g for 30 minutes , and the supernatant was precipitated hierarchically by ammonium sulfate .
to evaluate the protein concentration and hemagglutinating activity of each extract , all extracts were adjusted to the same volume using polyethylene glycol 20000 when necessary .
extraction and back extraction assays were performed by phase contact 1:1 ( v / v ) , stirred for 5 minutes and separated by centrifugation at 2292   g for 5 minutes following the method of nascimento et al . .  
single factor analysis was used to assess the effects of aqueous phase ph , extraction time and aqueous phase ion strength on reversed micelle extraction .  
bradford 's method   was used for protein quantification , using bovine serum albumin ( bsa ) as the standard .
the relative protein concentration of the eluted fractions was determined by measuring the absorbance at 280  nm .
serial two - fold dilutions of the lectin solution in microtiter v - plates ( 25  l ) was mixed with 25  l 2% chicken red blood cell suspension in saline ( ph 7.2 ) .
readings were recorded after about 30 minutes at room temperature , when the blank had fully sedimented .
the hemagglutination titer , defined as the reciprocal of the highest dilution exhibiting hemagglutination , was treated as one hemagglutination unit .
crude extract from reversed micelles was dialyzed against 0.1  m nacl in 0.02  m tris - hcl ( ph 8.0 ) , loaded on a deae sephadex a-50 column ( 2.6  cm  60  cm ) that had been equilibrated with the same buffer , and subjected to ion exchange chromatography .
the column was washed initially with 0.1  m nacl in 0.02  m tris - hcl ( ph 8.0 ) to remove proteins that had not specifically absorbed to the column , then washed with linear salt gradient elution .
fractions showing hemagglutinating activity were further purified by sieve chromatography on a sephadex g150 column in 0.15  m nacl in 0.02 m tris - hcl ( ph 8.0 ) .  
sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) was performed in accordance with the method of laemmli et al . , using a 15% separating and a 5% stacking gel .
powders ground from four chinese medicinal materials ( astragalus mongholicus , poria cocos , indigowood root ( isatis indigotica ) , and angelica sinensis ) were boiled in distilled water for 4 hours at   100c .
crude polysaccharide fractions were obtained by precipitation with three volumes of ethanol and desiccation in vacuo .
the precipitates were redissolved in distilled water and loaded on a d101 macroporous resin column ( 2.6  cm  60  cm ) to remove pigments .
the effluents were collected and polysaccharide fractions were quantitatively determined using a phenol - sulfuric acid assay   with glucose as the reference standard .   three kunming mice ( 2025  g ) were killed by cervical dislocation . the spleens were aseptically removed and pressed through a sterilized 100-mesh stainless steel sieve .
splenic lymphocytes were separated with lymphocyte separation medium and resuspended to a concentration of 2  10 cells / ml in rpmi 1640 culture medium supplemented with 10% fetal bovine serum , 100  units / ml penicillin and 100 g / ml streptomycin .
the suspensions ( 2  10  cells/90  l / well ) were seeded to a 96-well culture plate , and serial two - fold dilutions of red kidney bean lectin solution ( 0.78200  g / ml ) or chps solution ( 25400  g / ml ) in 10  l medium were added .
after incubation for 48 hours at   37c in humidified atmosphere containing 5% co2 , 10  l 0.5  mg / ml mtt was added , and the cells were incubated for another 4 hours under the same conditions . finally , 100  l dimethyl sulfoxide ( dmso ) was added to each well , oscillated for 1 minute , and the absorbance was measured at 570  nm .
the results were expressed as od values .   according to the mitogenic activity of red kidney bean lectin , an optimal lectin concentration ( 12.5  g / ml )
was chosen to be applied concurrently with the four chps at different concentrations ( 0 - 400   g / ml ) to detect whether mitogenic activity was enhanced .  
sixty kunming mice ( 2025  g ) were randomly divided into six groups , consisting of a saline control group , aps ( astragalus mongholicus polysaccharide ) group , lectin group , and lectin together with high , moderate , or low aps dosage group .
the mice were maintained in an air - conditioned animal room with an ambient temperature of 21c , 55% relative humidity , and 12 h/12 h light / dark cycle .
the animals were provided ad libitum with a basal diet ( institute of laboratory animal science , ingredients : protein 18% , fat 4% , fiber 5% , lysine 0.82% , calcium 1.01.8% , phosphorus 0.61.2% , and salt 0.30.8% ) and regularly refreshed tap water . the mice were injected intraperitoneally with 0.5  ml polysaccharide solution once per day for 5 days .
the lectin and high , moderate , or low aps dosage group received 12.5  mg / kg / bw lectin and either 100 , 50 , or 25  mg / kg / bw aps , respectively ; the aps group was given 100  mg / kg / bw aps ; the lectin group received 12.5  mg / kg / bw red kidney bean lectin ; and 0.5  ml saline was injected in the control group . on the seventh day a carbon particle clearance experiment was carried out , according to zhang , and the spleen index and thymus index were measured .
briefly , 0.05  ml/10 g / bw india ink was injected into each mouse through the vena caudalis , then 20  l blood was obtained from the vena orbitalis posterior 3 and 7 minutes after the injection .
the blood samples were placed in tubes with 2  ml 0.1% na2co3 , and od values were measured at 600  nm . the clearance index ( k ) , phagocytic index (  ) and immune organ index were calculated as follows , t2 and t1 means 7 minutes and 3 minutes respectively .  

( 1)k=(logod3logod7)t2t1,=body  weight(spleen  weight+liver  weight)k3 ,  immune  organ  index = immune  organ  weight  ( mg)body  weight  ( g ) . 

the extraction was optimal when the aqueous phase ph was 46 , and for back extraction , the optimal ph was 9 to 11 ( figure 1(a ) ) .
the highest lectin concentration was obtained with an extraction time of 46 minutes , beyond which the extraction rate decreased greatly , while the back extraction rate increased as the extraction time increased , but did not increase any further after 8 minutes extraction ( figure 1(b ) ) .
the ion strength of the aqueous phase had a major influence on reversed micelle extraction . at 0.05 
m nacl the aqueous and organic phases could not be detached , at 0.10  m nacl detachment was incomplete , at 0.15  m nacl complete detachment occurred and according to the curve the extraction effect was the best .
the regression equation of the bsa standard curve was y = 0.6476x0.0023   ( r = 0.9992 )   and the linear range was 0.21.0  mg / ml . the protein concentration of each extract was calculated based on the regression equation , and results showed that the reversed micelle extraction rate of degreased or undegreased ground bean was 7.23% and 6.25% , while those of traditional extraction methods were 4.27% , 5.49% , and 3.04% , respectively ( table 1 ) .
mean specific hemagglutinating activity of extracts obtained with the soak , degreasing , and homogenizing methods was 60 , 187 , and 84  titer / mg , respectively , whereas that of extracts obtained from degreased and undegreased red kidney bean powder with reversed micelles was 283 and 164 titer / mg , respectively ( table 1 ) .  
three fractions were obtained from ion exchange chromatography ( figure 2 ( a ) ) , among which fraction i did not show hemagglutinating activity , and the volume obtained from fraction iii was too small , so only fraction ii was collected , concentrated and loaded on a sephadex g-150 column for gel filtration .
fraction ii was resolved into a large fraction ( fraction iv ) and a very small fraction ( fraction v ) ( figure 2 ( b ) ) .
both fractions showed hemagglutinating activity , but only fraction iv was collected and dialyzed , then stored at 20c .
the purified lectin ( fraction iv in figure 2 ( b ) ) formed a single band with a molecular mass of about 30 kda in sds - page electrophoresis ( figure 3 ) , which confirmed the effectiveness of the purification method used .
the regression equation of the glucose standard curve was y = 0.0509x  0.0149 ( r = 0.9915 ) and the linear range was 210  g / ml . based on the standard curve , 3.65  g aps , 1.22  g pwp ( poria cocos polysaccharide ) ,
3.214  g irp ( indigowood root polysaccharide ) , and 3.55  g asp ( angelica sinensis polysaccharide ) were extracted from 300  g astragalus mongholicus , 200  g poria cocos , 300  g indigowood root , and 300  g angelica sinensis , respectively , and the carbohydrate concentration of each was 86% , 79% , 72% , and 83% , respectively ( table 2 ) .  
red kidney bean lectin significantly promoted proliferation of murine splenic lymphocytes at a final concentration of 6.25200  g / ml ( figure 4 ( a ) ) .
the four chps all showed mitogenic activity when applied alone , and their mitogenic activities were enhanced when applied concurrently with red kidney bean lectin , among which 50  g / ml aps with 12.5  g / ml red kidney bean lectin showed the highest mitogenic activity ( figure 4 ( b ) ) .
immune organ indices were calculated to evaluate the effects of aps and red kidney   bean lectin on mouse immune functions .
it was found that 100  mg / kg / bw aps with 12.5  mg / kg / bw red kidney bean lectin increased the mouse spleen index , but there were no significant differences in the thymus index between the treated groups and control groups ( table 3 ) . the aps group and concurrent application of aps with moderate and high doses of red kidney bean lectin increased the phagocytosis activity of macrophages .
lectins possess many bioactivities that may have important practical applications , but the high price resulting from the low extraction rate restricts practical application of lectins . in this study a novel method , reversed micelles , was used to extract red kidney bean lectins .
the interface between the water and apolar liquid is stabilized by a monolayer of surfactant molecules , the polar heads of which are in contact with the aqueous phase , whereas the hydrocarbon radicals are directed inside the oil phase .
reverse micellar systems have been utilized for extraction and purification of proteins since the 1990s [ 26 , 27 ] .
nascimento et al .   used reverse micelles to extract affinity - purified lectin from cratylia mollis seeds , of which the extraction and back extraction rate were both 100% . in the present study ,
the optimal conditions were found to be extraction at ph 46 and back extraction at ph 911 , an aqueous phase ionic strength of 0.15  m nacl , extraction for 46 minutes and back extraction for 8 minutes .
extraction and back extraction of reversed micelles is very fast and both completed in a few minutes .
compared to the tedious operations of traditional extraction methods , reversed micelles were shown to have much greater efficiency .
the efficacy of degreasing with both traditional methods and reversed micelles was investigated , and degreasing was demonstrated to be conducive to the separation of red kidney bean lectin . reversed micelles resulted in a higher extraction rate and higher hemagglutinating activity than the three traditional methods , and the protein extraction rate from degreased bean powder with reversed micelles was 31.60% higher than that with the degreasing method ( the best of the three traditional methods ) .
the total hemagglutinating activity of reversed micelles was 99.80% higher than that of the degreasing method ( 10.23  10 and 5.12  10 , resp . ) .
therefore , reversed micelles were considered to be a superior method to extract lectins .  
methods for purifying lectins differ from one to another according to the source of the lectins , but classical protein purification methods , such as salting out and different chromatographic methods [ 6 , 28 , 29 ] , are still the basic methods used to purify lectins . in this
study the purification procedure involved ion - exchange chromatography on deae sephadex a-50 and sieve chromatography on sephadex g150 columns .
the purified lectin represented by fraction iv appeared as a single band with a molecular mass of 30  kda in sds - page , which demonstrated that the purification strategies were effective .  
mitogenic activity has been identified in many leguminous species , such as knife beans   and anasazi beans .
phytohemagglutinin ( pha ; lectin extracted from phaseolus vulgaris ) and concanavalin a ( cona ; lectin extracted from canavalia gladiata ) are the two main mitogens used in cell proliferation experiments in vitro .
concentrations of pha used in cell proliferation assays in vitro differ according to the kidney bean cultivar and breeding location , the purpose and method of the assay , and source of pha ; the ultimate concentration of pha obtained from sigma has varied from 0.04  g / ml   to 1  g / ml , 5  g / ml [ 33 , 34 ] , 10  g / ml [ 3538 ] , 14  g / ml , 20  g / ml , and 25  g / ml , and that from shanghai yihua   and tianjin guangfu   is 10  g / ml .
this article demonstrated that red kidney bean lectin can notably stimulate splenic lymphocyte proliferation at concentrations from 6.5 to 200  g / ml .
thus , compared with commercial pha , this lectin has potential practical applications as a mitogen .  

astragalus mongholicus , poria cocos , poria cocos , indigowood root and angelica sinensis are four well - known traditional east asian medicinal plants , polysaccharides of which are important ingredients with many beneficial functions [ 4446 ] .
a lot of chps have mitogenic activity , such as astragalus polysaccharide , irp and chinese yam polysaccharide [ 45 , 47 ] .
mitogenic activities of polysaccharides from these four plants were studied in the presence or absence of red kidney bean lectin .
all four polysaccharides were shown to possess mitogenic activity , and the activity was enhanced when the chps were applied concurrently with red kidney bean lectin , among which joint application of aps with red kidney bean lectin exhibited the strongest mitogenic activity .
herbal polysaccharides exert immunoregulatory effects by different and complex mechanisms , including modulating the th1/th2 ratio , phagocytosis activity of macrophages , of nk cell activity and secretion of cytokines .
mitogenic activities of polysaccharides have been studied for decades , but the mechanisms are still unclear . in this
study the mitogenic activities of aps , irp , and pwp were suppressed at a high concentration , which has been reported in previous studies [ 48 , 49 ] .
reasons for this phenomenon might be the proliferation of ts cells or presence of cytotoxic contaminants in the polysaccharides .   
macrophages are an important component of host defenses against viral infection by inhibiting intracellular replication of viruses and by killing virus - infected cells .
when activated , a variety of oxygen or nitrogen intermediates and cytokines are released from macrophages and participate in various important biological functions , such as anti - inflammatory and anti - tumor activities [ 44 , 51 , 52 ] .
many herbal polysaccharides can enhance phagocytic activity of macrophages [ 44 , 53 , 54 ] . in this study aps augmented the phagocytic activity of macrophages , which increased 9.07% when aps and red kidney bean lectin were applied concurrently .
joint application of aps and red kidney bean lectin also increased the spleen index , whereas application of aps and red kidney bean lectin alone did not .
thus , enhancements in effects were apparent when herbal polysaccharides were applied concurrently with red kidney bean lectin , which is a very important finding considering the high cost and time - consuming nature of the extraction procedures .
 
                   reversed micelles were used to extract lectin from red kidney beans and factors affecting reverse micellar systems ( ph value , ionic strength and extraction time ) were studied . 
 the optimal conditions were extraction at ph 46 , back extraction at ph 911 , ion strength at 0.15  m nacl , extraction for 46 minutes and back extraction for 8 minutes . 
 the reverse micellar system was compared with traditional extraction methods and demonstrated to be a time - saving method for the extraction of red kidney bean lectin . 
 mitogenic activity of the lectin was reasonably good compared with commercial phytohemagglutinin ( extracted from phaseolus vulgaris ) mitogenic properties of the lectin were enhanced when four chinese herbal polysaccharides were applied concurrently , among which 50  g / ml   astragalus mongholicus polysaccharides ( aps ) with 12.5  g / ml red kidney bean lectin yielded the highest mitogenic activity and 100  mg / kg / bw   aps with 12.5  mg / kg / bw red kidney bean lectin elevated mouse nonspecific immunity . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion