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A New Lectin from the Tuberous Rhizome of Kaempferia rotunda: Isolation, Characterization, Antibacterial and Antiproliferative Activities

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A New Lectin from the Tuberous Rhizome of Kaempferia rotunda: Isolation, Characterization, Antibacterial and Antiproliferative Activities
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  1140  Protein & Peptide Letters, 2011, 18, 1140-1149   0929-8665/11 $58.00+.00 © 2011 Bentham Science Publishers A New Lectin from the Tuberous Rhizome of  Kaempferia rotunda : Isola-tion, Characterization, Antibacterial and Antiproliferative Activities Syed Rashel Kabir  1  *, Md. Amir Hossen 1 , Md. Abu Zubair  2 , Md. Jahangir Alom 1 , Md. Farhadul Islam 1 , Md. Anowar Hossain 1  and Yoshinobu Kimura 3   1  Department of Biochemistry and Molecular Biology, Faculty of Science, Rajshahi University, Rajshahi-6205, Bangla-desh, 2  Department of Food Technology and Nutritional Science, Mawlana Bhashani Science and Technology University, Santosh, Tangail-1902, Bangladesh, 3  Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan Abstract:  A lectin (designated as KRL) was purified from the extracts of Kaempferia rotunda  Linn. tuberous rhizome by glucose-sepharose affinity chromatography. KRL was determined to be a 29.0±1.0 kDa polypeptide by SDS-PAGE under  both reducing and non-reducing conditions. KRL was a divalent ion dependent glycoprotein with 4% neutral sugar which agglutinated different groups of human blood cells. Methyl-  -D-mannopyranoside, D-mannose and methyl-  -D-glucopyranoside were the most potent inhibitors. N-terminal sequence of KRL showed similarity to some man-nose/glucose specific lectins but the main differences with their molecular masses and sugar content. KRL lost its activity markedly in the presence of denaturants and exhibited high agglutination activity from pH 6.0 to 8.2 and temperature 30 to 60ºC. The lectin   showed toxicity against brine shrimp nauplii with the LC 50  value of 18±6  g/ml and strong agglutina-tion activity against seven    pathogenic bacteria. KRL inhibited the growth of six bacteria partially and did not show anti-fungal activity.   In addition, antiproliferative activity against Ehrlich ascites carcinoma (EAC) cells showed 51% and 67% inhibition in vivo  in mice administered 1.25 mg/kg/day and 2.5 mg/kg/day of KRL respectively by injection for five days. Keywords: Antibacteria, antiproliferative, bacterial agglutination, lectin, lethality assay, mannose. INTRODUCTION The Kaempferia rotunda linn. tuberous rhizome locally known as ‘Misrydana’ belongs to the family of Zingib-eraceae, and is a fragrant aromatic herb with a tuberous rhi-zome distributed throughout India and some districts in Bangladesh. There are 26 genera and more than 300 species of this plant in Thailand [1]. The tuberous rhizome Kaempferia rotunda  linn. has been used as food and tradi-tional medicinal plant for the wide spectrum of illness such as ulcer, gastrointestinal disorders etc. Many phytochemicals have been isolated from this tuberous rhizome with different  bioactivities [2-6] Lectins are a group of proteins found in all types of living organisms, either in soluble or in membrane-bound form that recognizes specific carbohydrate structures and thereby ag-glutinate cells by binding to cell-surface glycoproteins and glycoconjugates [7]. On the basis of structural and evolu-tionary development, most of these plant lectins have been classified as legume lectins, chitin-binding proteins, type 2 ribosome-inactivating proteins, monocot mannose-binding lectins, amaranthins, cucurbitaceae phloem lectins and  jacalin-related lectins [8]. The rich sources of lectins in  plants are particularly their organs such as seeds, tubers,  bulbs, rhizomes, bark etc. and they have attracted great inter-est on according of their various biological activities, such *Address correspondence to this author at the Department of Biochemistry and Molecular Biology, Faculty of Science, Rajshahi University, Rajshahi-6205, Bangladesh; Tel: +880-721-750041/4109; Fax: 880-721-750064; E-mails: rashelkabir@gmail.com; rashelkabir@ru.ac.bd as cell agglutination [1], antifungal [9], antiviral [10] and antiproliferative activities [11-12]. Specially mannose and  N  -acetyl-D-glucosamine-binding lectins are considered bio-logically important defense proteins, because these sugars are widely distributed in micro-organisms, insects and ani-mals [13-14]. Although different sugar specific lectins were  purified from rhizomes of different sources e.g., mannose specific lectins form Ophiopogon japonicus  and Smilax glabra Roxb [10, 15], mannose/maltose specific lectin form Calystegia sepium [16], N-acetyl-D-glucosamine specific lectin from  Arundo donax  [12], chitin-binding lectin from Setcreasea purpurea  [17], but till now purification of lectin was not reported from tuberous rhizomes of Kaempferia ro-tunda . In this manuscript first time we are reporting the puri-fication and characterization of a new lectin from the me-dicinal plant Kaempferia rotunda  with antibacterial and anti- proliferative activities. MATERIALS AND METHODS Materials and Sample Sepharose 4B was procured from Fluka (Sweden). All other chemical/reagents were of the highest grades commer-cially available. The tuberous rhizome of Kaempferia ro-tunda were collected from the local market and stored at 4ºC. Purification of Protein   Kaempferia rotunda  rhizome was homogenized in 10 mM Tris-HCl buffer saline (Tris-HCl buffer containing 150   A New Lectin from Tuberous Rhizome of Kaempferia rotunda Protein & Peptide Letters, 2011  , Vol. 18, No. 11 1141   mM NaCl), pH 8.2 (500 ml buffer for 100 g tuber). The ho-mogenate was centrifuged at 8,200 g for 30 min and the su- pernatant was dialyzed against distilled water and then against 10 mM Tris-HCl buffer, pH 8.2. After dialysis, the crude sample was centrifuged at 8,200 g for 15 min and then the supernatant was collected and incubated with glucose-sepharose (previously equilibrated with the same buffer con-taining 10 mM CaCl 2 ) at 4ºC with occasional shaking. The column was packed with 20 ml of glucose-sepharose (glu-cose linked to epichlorohydrin-activated sepharose-4B) and the unbound protein was washed out with 10 mM Tris-HCl  buffer saline, pH 8.2 (containing 1 mM CaCl 2 ). Bound pro-teins were first eluted by the same buffer saline containing 0.6 M glucose and then by 10 mM sodium acetate buffer saline, pH 4.6 (containing 5 mM ethylenediaminetetraacetic acid, EDTA). The pH of the eluted fraction was adjusted around 7.0 by the addition of 1 M Tris-HCl buffer, pH 8.2 and then dialyzed against distilled water (DW) and 10 mM Tris-HCl buffer, pH 8.2. The purity was checked by using SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) in 16% (w/v) polyacrylamide gel as de-scribed by Laemmli [18]. The existence of subunits was checked on the same gel in the presence and absence of 2-mercaptoethanol. The protein elution profiles were moni-tored at 280 nm. The purified lectin was designated as Kaempferia rotunda  lectin  ( KRL). Molecular Mass Determination The molecular mass of the purified protein was deter-mined by SDS-PAGE using 16% (w/v) polyacrylamide gel. Bovine serum albumin (67 kDa), Ovalbumin (45 kDa), Car- bonic anhydrase (29 kDa), Trypsin inhibitor (20 kDa) and Lysozyme (14.6 kDa) were used as marker proteins. Erythrocytes Blood group specificity was tested using different types of human blood groups (A, B, O and AB) collected from four donors and the chicken blood was collected from a slaughter house. All the blood samples were collected in saline and centrifuged at 1,027 g for 10 min. The erythrocyte  pellets (RBC) were washed thrice and resuspended in the same saline to prepare a 2% suspension. Hemagglutination Assay The hemagglutination assay was performed in 96-well microtiter U-bottomed plates in a final volume of 100  l, which containing 50  l (0.4 mg/ml) of protein solution seri-ally diluted with equal amount of hemagglutination buffer (20 mM Tris-HCl buffer saline, pH 7.8 containing 10 mM CaCl 2 ) and 50  l of 2% suspension of albino rat erythrocytes  previously washed with saline. After a gentle shaking, the  plate was kept at room temperature (25 o C) for 30 min. The visual agglutination titer of the maximum dilution giving  positive agglutination was recorded. Inhibition of hemagglu-tinating activity was examined by adding serial dilutions of the following sugars: D-mannose, maltose, D-glucose, D-galactose, L-arabinose, D-xylose, L-rhamnose, D-melibiose, methyl-  -D-mannopyranoside, methyl-  -D-glucopyranoside,  N-acetyl-D-glucosamine, methyl-  -D-glucopyranoside, methyl-  -D-galactopyranoside, D-glucosamine-HCl at the final concentration of 200 mM; 4-nitrophenyl-  -D-galacto- pyranoside, 4-nitrophenyl-  -D-mannopyranoside and 2-nitrophenyl-  -D-galactopyranoside at the final concentration of 25 mM. Effect of Temperature and pH on Hemagglutination Ac-tivity To examine the thermo stability, KRL (0.4 mg/ml in Tris-HCl buffer saline, pH 7.8) was heated in a water bath for 30 min at different temperatures from 30-90ºC and cooled to room temperature. Then 50 μ l of KRL was serially diluted with an equal amount of hemagglutination buffer, pH 7.8 and the hemagglutination titer was performed as ex- plained above. The non-heated lectin sample was used as a control, which denoted 100% activity. The pH stability was determined by incubating KRL solutions (0.4 mg/ml) against different buffer saline (pH value ranging from 3 to 12.5) for 8 h at room temperature. Then the lectin solutions were dia-lyzed against 20 mM Tris-HCl buffer saline, pH 7.8 for 12 h. The following buffers were used for pH stability assay: 0.1 M sodium acetate (pH 3-6), 0.1 M phosphate (pH 7.0), 0.1 M Tris-HCl (pH 8.2) and 0.1 M glycin-NaOH (pH 9-12.5). Estimation of Protein and Sugar Content Protein was estimated by the Lowry method [19] using lipid-free BSA as the standard. The sugar content of KRL was determined according to the phenol-sulfuric acid method [20] using D-glucose as standard. N-terminal Sequence Determination From SDS-PAGE, the lectin was transferred to a poly-vinylidene difluoride (PVDF) membrane and the band corre-sponding to the lectin was then excised from the membrane.  N-terminal sequence was determined by Edman degradation using an amino acid analyzer and the N-terminal sequence homology was analyzed using the BLAST (http://blast.ncbi.nlm.nih.gov) database search and the mo-lecular mass of the homologous lectins were calculated from the full amino acids sequences by using the following web site, http://proteome.gs.washington.edu/cgi-bin/aa_calc.pl. Effect of Detergents and Metal ions on Lectin-Induced Hemagglutination Activity For detecting the effect of denaturants, KRL (0.2 mg/ml) in 0.1 M Tris-HCl buffer saline was incubated at room tem- perature with 0.5, 1, 2 and 4 M of Urea; 0.5, 1, 2 and 4 of Guanidine-HCl for 2 h. KRL in the same buffer without de-naturants was used as a control and its activity was consid-ered as 100%. To determine the dependency of hemaggluti-nation activity on divalent cations, KRL was incubated with 100 mM EDTA (pH 8.0) for 2 h at room temperature. Then KRL solution was dialyzed against 20 mM Tris-HCl buffer saline, pH 7.8 for 12 h at 4ºC and subjected to hemagglutina-tion assay in the presence and absence of 10 m M of Ba 2+ , Ca 2+ , Mn 2+ and Mg 2+ in the hemagglutination buffers. Bacterial Agglutinating Activity The bacterial agglutinating activity was performed by using  Bacillus cereus, Bacillus subtilis, Bacillus megaterium,  1142  Protein & Peptide Letters, 2011  , Vol. 18, No. 11 Kabir et al. Sarcina lutea, Escherichia coli, Shigella shiga, Shigella dys-enteriae, Shigella sonnei, Salmonella typhi and Klebsiella sp . Bacteria were grown at 37ºC overnight in nutrient broths (liquid nutrient medium), then the bacteria were collected by centrifugation at 1,027 g for 5 min and washed with 10 mM Tris-HCl buffer saline, pH 7.8 and re-suspended in the same  buffer with a turbidity of 2.0 at A 640 . 50  l of each bacterial suspension was mixed with a serial dilution of KRL (0.3 mg/ml) to a final volume of 100  l in 96-well microtiter  plates. The plates were agitated for 2 min and the mixtures were kept at room temperature for 60 min. Finally, the bacte-rial agglutinating activity was monitored by a light micro-scope. Bacterial agglutination inhibition was studied above the method in the presence of 0.4 mM methyl-  -D-mannopyranoside. Brine Shrimp Nauplii Lethality Assay To study the lethality assay, 10 brine shrimp nauplii (  Artemia salina L ) in artificial sea water was added to differ-ent vials and then 2 mg/ml of KRL (in the presence and ab-sence of 0.4 mM methyl-  -D-mannopyranoside in sea water) was added to each vial at the final concentration of 1.25, 2.5, 5, 10, 15 and 20  g/ml and finally the volume of each vial was adjusted to 4 ml by the addition of artificial sea water. Artificial sea water was prepared by dissolving 38 g of NaCl in 1 liter of DW and the pH was adjusted to 7.0 by the addi-tion of sodium tetraborate. All tests were performed at 30ºC, under a continuous light regime. Three replicate were used for each experiment. From this data, the percentage of mor-tality of the nauplii was calculated for each concentration and the LC 50  values were determined using Probit analysis as described by Finney [21]. Bacterial Growth Inhibition The bacterial growth inhibition was performed by meas-uring the bacterial nutrient broths (liquid nutrient medium) in the presence and absence of different concentration (38  g/ml - 600  g/ml)   of KRL by using a titer plate reader at A 630 . Eight bacteria (  Bacillus cereus ,  Bacillus subtilis ,  Bacil-lus megaterium ,  Sarcina lutea ,  Shigella shiga ,  Shigella dys-enteriae ,  Shigella sonnei and   Klebsiella sp . ) were used for this study. Bacteria were grown at 37ºC overnight in nutrient  broths and the absorbance was adjusted with liquid nutrient medium to 0.18-0.2 at A 630 . Then 1 M of CaCl 2  was added to each bacterial media to the final concentration of 1 mM. In order to check the bacterial growth inhibition in the presence of sugar, 0.2 M of methyl-  -D-mannopyranoside was added in each bacterial media to the final concentration of 0.4 mM. 50  l of each bacterial suspension was mixed with the serial dilution of KRL to a final volume of 100  l in 96-well mi-crotiter plates. Three wells without lectin for each bacterium were used as control. The plates were agitated by using tem- perature controlled titer plate shaker at 28ºC and the reading was taken after 8 h at A 630 . Finally, the percent of bacterial growth inhibition in the presence of KRL was determined according to the formula: % inhibition = {(Absorbance of control - Absorbance of test) /Absorbance of control}   100 Antifungal Assay Antifungal activities of KRL were performed using ster-ile-petri dishes (70   7 mm) containing 10 ml potato dex-trose agar. The fungal mycelia were placed over the solid  potato dextrose agar and the sterile filter paper discs (5 mm in diameter) were distributed over the plates. 20  l and 10  l of KRL solutions (100  g/ml) in 10 mM Tris-HCl buffer saline, pH 8.0 (containing 2 mM CaCl 2 ) and the buffer (as  blank) were applied on the paper discs of the petri dishes. Incubation of the petri dishes were carried out at 30ºC until the mycelial growth had enveloped the dishes and formed crescents of inhibition around the discs with KRL. Three fungal species ( Candida albicans ,  Aspergillus niger  ,  Fusarium vasinfectum ) and one fungal genus (  Mucor   sp . ) were examined in this assay. Determination of Ehrlich ascites carcinoma (EAC) Cell Growth Inhibition The Ehrlich ascites carcinoma cells were propagated in-traperitonealy in mice in our departmental research labora-tory biweekly. EAC cells collected from a donor swiss al- bino mouse bearing 6-7 days old ascites tumors were diluted with the normal saline with an adjustment of 3 X 10 6  cells/ml and the cells were counted by haemocytometer. The viability of tumor cells was observed by trypan blue dye (0.4%) ex-clusion assay. Tumor cells showing above 90% viability were injected (0.1 ml) intraperitonealy to each swiss albino mouse. After 24 hours the mice were randomly distributed into three groups with at least five mice per group. Two groups of mice were treated intraperitonealy for five days with KRL at a concentration of 2.5 mg/kg/day (50  g/mouse/day) and 1.25 mg/kg/day (25  g/mouse/day) and the remaining group was used as the control. Mice in each group were sacrificed on day six; the total intraperitoneal tumor cells were harvested by normal saline and counted by a haemocytometer. The total numbers of viable cells in every mouse of the treated groups were compared with those of controls (EAC treated only). Percent of inhibition was calcu-lated by using the following formula: % of inhibition = 100 – {(cells from KRL treated mice / cells from control mice)    100}. RESULT Purification and Molecular Mass Determination A lectin was purified from the rhizome of   Kaempferia rotunda  crude extract by the affinity chromatography on glucose-sepharose column. Protein was eluted first by sugar and then by acetate buffer as shown in Fig. 1A . The sugar eluted fraction did not show any hemagglutination activity, whereas the sodium acetate eluted fraction showed hemag-glutination activity and migrated on SDS-PAGE as a single  band with an apparent molecular mass of 29.0±1.0 kDa in the presence and absence of 2-mercaptoethanol (Fig. 1B ). About 15 mg of KRL obtained from 500 g of   Kaempferia rotunda  tuberous rhizome and the purification procedure was summarized in Table 1 .   A New Lectin from Tuberous Rhizome of Kaempferia rotunda Protein & Peptide Letters, 2011  , Vol. 18, No. 11 1143   Hemagglutination Assay and Carbohydrate Specificity of the Purified Proteins The minimum agglutinating activity of the KRL was found to be 3 μ g/ml for rat erythrocyte, 6 μ g/ml for each group of human erythrocytes (A, B, O and AB) and did not show any agglutination activity against chicken blood. The carbohydrate-binding specificity was evaluated by inhibiting the agglutination of rat erythrocytes using different sugars. The best inhibitor for KRL was found to be methyl-  -D-mannopyranoside followed by D-mannose and methyl-  -D-glucopyranoside as presented in Table 2 . Fraction A did not show any agglutination activity against the said blood cells. Protein and Sugar Analysis KRL gave orange yellow color when it was subjected to the phenol-sulfuric acid method and the neutral sugar content of KRL was estimated to be 4%. The absorbance of 1.0 at 280 nm for KRL corresponded to 0.6 mg of protein. N-Terminal Sequence Determination The first seven residues of the N-terminal sequence of KRL were determined to be ADTIVAV. The sequence showed similarity to the first seven residues with some other lectins and the calculated molecular masses were 25 to 25.6 kDa as shown in Table 3 . Effect of Temperature and pH on Hemagglutination Ac-tivity Thermal inactivation of KRL was investigated by incu- bating the lectin at different temperatures for 30 min and assaying the hemagglutination activity. Highest lectin activ-ity was observed between 30-60ºC. The lectin lost its activity 50 and 97% at 70 and 80ºC respectively and completely at 90ºC. KRL was observed to be stable between the pH 3.0 to 12.5 and showed the maximum activity from the pH 6.0 to 8.2. At pH 3.0 to 4.0 lectin lost its activity 87.5%, whereas at  pH 9.0 to 12.5 lost of 75% activity were found. Fig. (1). ( A ) Affinity chromatography of KRL. Crude protein was applied to a glucose-sepharose column (20ml) previously equilibrated with 10 mM Tris-HCl buffer saline, pH 8.2. Proteins were eluted with the same buffer saline containing 0.6 M glucose and then 10mM Sodium acetate buffer saline, pH 4.6 containing 5mM EDTA. The elution profiles were monitored at 280 nm. Fractions (2.5 ml/tube) were collected at 1 ml/min flow rate. ( B )   SDS-PAGE of KRL on 16% polyacrylamide gel. Lane 1: KRL in the presence of 2-mercaptoethanol; lane 2: KRL in the absence of 2-mercaptoethanol; lane 3: Marker proteins. Table 1. Purification Scheme of KRL Purification Steps Total Protein (mg) Total Activity (Titer/mg   10 3 ) Recovery of Activity (%) Purification Fold Yields (%) Crude 2500 800000 100 1 100 Affinity Chromatography on glucose-sepharose 15 192000 24 40 0.6 00.10.20.30.40.50.6    A   b  s  o  r   b  a  n  c  e  a   t   2   8   0  n  m    T  r   i  s  -   H   C   l   b  u   f   f  e  r  s  a   l   i  n  e  w   i   t   h   0 .   6   M   g   l  u  c  o  s  e  AB    S  o   d   i  u  m   a  c  e   t  a   t  e   b  u   f   f  e  r 0 5 10 15 20 25 Fractions (2.5 ml/tube) 6745292014.6 1 2 3     1144  Protein & Peptide Letters, 2011  , Vol. 18, No. 11 Kabir et al. Table 2. Hemagglutination Inhibition of KRL by Mono- and Oligosaccharides Sugar Minimum Inhibitory Con-centration (mM) Methyl-  -D-mannopyranoside 0.4 D-mannose 1.6 Methyl-  -D-glucopyranoside 1.6 Maltose 3.2  N-Acetyl-D-glucosamine 12.5 D-glucose 25 Methyl-  -D-glucopyranoside 25 D-galactose NI L-arabinose NI D-glucosamine-HCl NI D-xylose NI L-rhamnose NI D-melibiose NI Methyl-  -D-galactopyranoside NI 4-Nitrophenyl-  -D-galactopyranoside *NI 4-Nitrophenyl-  -D-mannopyranoside *NI 2-Nitrophenyl-  -D-galactopyranoside *NI  NI, no inhibition at 200 mM of sugars. *NI, no inhibition at 25 mM of sugars. Effect of Denaturants and Divalent Ions on KRL Activity The lectin activities decreased to half in the presence of 0.5 M of urea, further the activities decreased up to 87% which was constant at each of 1, 2 and 4 M of urea. On the other hand, the activity of KRL declined to 97% at 0.5 M of guanidine-HCl and lost the activity completely at 1 M or above the concentration as compared with the control. Fur-thermore, EDTA treated KRL did not show any heagglutina-tion activity in the absence of divalent ions in the hemagglu-tination buffer. But after the addition of 10 mM of Ca 2+ and Mg 2+  the lectin recovered 100% of its activity, whereas in the  presence of Ba 2+  and Mn 2+  the activity was recovered 50% only Bacterial Agglutinating Assay   In absence of sugar, KRL agglutinated both gram- positive bacteria (  Bacillus subtilis, Bacillus cereus, Bacillus megaterium and Sarcina lutea )   and   gram - negative    bacteria  (Klebsiella species, Escherichia coli, Shigella sonnei and  Salmonella typhi ). Whereas in the presence of sugar, KRL did not show any agglutination activity against the said bac-teria. The minimum concentration of KRL solution needed for the agglutination of each bacterium was summarized in Table 4 . Brine Shrimp Nauplii Lethality Assay   In the presence and absence of sugar, the toxic effect of KRL against brine shrimp nauplii was determined. In the  presence of sugar, KRL did not show any toxic effect but in the absence of sugar the mortality rate of brine shrimp nau- plii had gone up with the increased of concentration of the lectin as shown in Fig. 2  and the LC 50  value for KRL was calculated to be 18±6 μ g/ml. Bacterial Growth Inhibition   The presence of KRL inhibited the growth of   Shigella sonnei ,  Bacillus cereus ,  Bacillus subtilis ,  Bacillus megate-rium , Klebsiella sp. and Sarcina lutea . When the concentra-tion of KRL was increased, the growth of  Bacillus cereus  and  Bacillus subtilis  became decreased. The growth inhibi-tion remained almost constant for Shigella sonnei  and  Bacil-lus megaterium  at certain concentration range but  Klebsiella sp. and Sarcina lutea  were inhibited only at high doses of lectin as shown in Fig. 3 . On the other hand, the presence of KRL did not affect the growth of   Shigella shiga and Shigella dysenteriae . When the experiment was done in the presence of sugar bacterial growth inhibition was not observed. Antifungal Activity   KRL did not show antifungal activity against Candida albicans, Aspergillus niger, Fusarium vasinfectum and  Mu-cor sp . EAC Cell Growth Inhibition KRL effectively inhibited the proliferation of EAC cells. 51% inhibition was obtained at 1.25 mg/Kg/day but 67% inhibition was observed at 2.5 mg/Kg/day as shown in Fig. 4 . DISCUSSION A lectin was purified from Kaempferia rotunda tuberous rhizome s  designated as KRL. The lectin strongly aggluti-nated rat erythrocytes with the minimum concentration of 3 μ g/ml and did not show any human blood group specificity as it agglutinated A, B, O and AB blood groups. Hemagglu-tination inhibition studies revealed that methyl-  -D-manno- pyranoside, D-mannose and Methyl-  -D-glucopyranoside were the most potent inhibitor for KRL and the minimum inhibitory concentration was 0.4, 1.6 and 1.6 mM respec-tively. The first seven residues of the N-terminal amino acids of KRL were determined as ADTIVAV which was identical with the lectins from Canavalia ensiformis  [22], Canavalia virosa  [23], Canavalia brasiliensis [24], Canavalia gladiata [25],  Dioclea lehmanni [26] and some other mannose spe-cific seed lectins as showed in Table 3 . The purified lectin migrated with single band in SDS-PAGE and the apparent molecular weight was 29.0±1.0 kDa both in the presence and absence of  -mercaptoethanol. On the other hand, Concana-valin-A from Canavalia ensiformis yielded three major  bands in SDS-PAGE [27]. This behavior was similar to all lectins isolated from  Dioclea violacea [28],  Canavalia vi-rosa , Canavalia maritime ,  Canavalia   gladiata  [29],
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