Complex of zinc and lectins from seeds of Vigna radiata as potential anti-diabetic agent Chhaya Gadgoli 1, *, Lalit Sali, Mahesh Abhyankar, Prachi Pathak 1 1 Saraswathi Visya Bhavan’s College of Pharmacy, Sankara Nagar Dombivli (E) 421204, Maharashtra,India . chhayahgadgoli@gmail.com 1
Complex of zinc and lectins from seeds of Vigna radiata as potential anti-diabetic agent Crude Macerati drug on Centrifugation Dialysis Precipitatio n SDS- PAGE Gel Western Affinit Blot y Zn Lectin Complex Invivo study 2 2
Abstract The V. radiata seed was found to contain galactose specific lectin. The MBL-I (Mung Bean LectinI ) may be a tetrameric protein with molecular weight 160-180 kDa and may be composed of identical or nearly identical relative subunits of molecular weight of 45-50 kDa. Amino acid analysis of purified Mung bean lectin by reverse phase HPLC revealed that it contains Glutamate in highest proportion followed by Aspartate and also contains Histidine, which indicates it has good zinc binding potential. Binding of the lectins with zinc improve overall stability and efficacy of the lectins. The antidiabetic activity was evaluated in Wistar rats using alloxan induced diabetic model and the studies indicated significant (P<0.001) reduction in elevated sugar levels. A probable mechanism of antidiabetic action may be insulinomimetic, since it was found to bind with insulin antibodies in Western Blotting Analysis. The lectin obtained from V. radiata seed and zinc lectin complex has good potential to be explored as a safe natural antidiabetic agent acting with controlled reduction in blood glucose levels. These findings indicate that the mungbean lectins and zinc lectins complexes have tremendous medicinal potential as a herbal antidiabetic drug. Keywords: Lectins; Vigna radiata; antidiabetic activity 3
Introduction Lectins from Vigna radiata are sugar-binding proteins that agglutinate cells They have promising results in antidiabetic studies Insulin-Zinc complex gives better effect and stability. Based on this, the aim of this research was to investigate antidiabetic activity of lectins from Vigna radiata complexed with zinc in alloxan induced diabetic wister albino rats. 4
Results and Discussion C HARACTERIZATION OF LECTINS HEAMAGGLUTINATION ACTIVITY : Performed by Standard dilution technique using rabbit erythrocyte suspension. The effect of sugars, pH and temperature on hemagglutination activity was studied. ASSAY OF α AND β GALACTOSIDASES : α Galactosidase activity is more as compared to the β Galactosidase in V.radiata samples . 5
GEL ELECTROPHORESIS SDS-PAGE data revealed the Native-PAGE data revealed the presence of a single sharp in presence of a single sharp in the region of 45-50 kDa the region of 160-180 kDa. All these data indicate that the V. radiata seed lectin may be a tetrameric protein with a relative molecular weight of about 160-180 kDa and identical or nearly identical subunits of 45-50 kDa. 6
WESTERN BLOTTING Western Blotting analysis shows that purified lectin has fewer insulin binding sites than freeze dried lectins but may have the mechanism of action as that of insulin in lowering the elevated blood glucose levels. 7
P REPARATION OF ZINC LECTINS COMPLEX AND ITS EVALUATION Binding pattern of Zinc with the lectins The maximum % w/w binding of zinc with the lectins was found out to be 1.178 % w/w at 1000 μg /ml of zinc chloride (0.47996 mg/ml of Zn +2 ) 8
TEM & DSC Analysis of Lectins and Zinc Lectin Complex Avg Particle Size Avg Particle Size 1.259µm 1.326µm Spray Dried Lectins Spray Dried Lectins and zinc complex M.P 129.3 °C ∆Hcal:183.73mj/mg M.P 118.7 °C ∆Hcal:227.49mj/mg 9
IN-VIVO EVALUATION OF LECTINS & ZINC LECTIN COMPLEX Acute toxicity studies (as per OECD 425) Treatment group Number of Mortality Dose (Female Wistar rats) animals observed 2000 mg/kg 06 No mortality Saline suspension of b.w.s.c. lyophilized crude lectins 2000 mg/kg 06 No mortality b.w.p.o. 2000 mg/kg Saline suspension of Zinc 06 No mortality b.w.s.c. freeze dried lectins 2000 mg/kg complex 06 No mortality b.w.p.o. Saline suspension of crude lectins & Zn lectin complex is practically safe & its LD50 (MTD) is higher than 2000 mg/kg 10
ORAL GLUCOSE TOLERANCE TEST (OGTT) 200 180 Normal Control Blood Sugar Levels mg/dl 160 Glucose Control 140 120 Glibeclamide (Standard) 100 150mg/kg bw.s.c (zn 80 lectin complex) 60 250mg/kg bwp.o (zn lectin complex) 40 300 mg/kg bw s.c 20 (lectins) 0 0 30 60 90 120 240 +Glucose Time in (Minutes) The risk of hypoglycemia observed in Glibenclamide treated group is not observed in lectins as well as Zn lectins complex treated group. 11
EFFECT OF FREEZE DRIED LECTINS & ZINC LECTIN COMPLEX ON SERUM GLUCOSE LEVELS 350 Normal Control Serum Glucose Levels mg/dl 300 Diabetic Control 250 Glibeclamide (Standard) 200 250mg/kg bw.s.c (lectins) 150 350mg/kg bw.p.o (lectins) 100 150mg/kg bw.s.c (Zn lectin complex) 50 150mg/kg bw.p.o (Zn lectin complex) 0 Day 0 Day 1 Day 7 Day 14 Treatment Period (Days) Lowering of the serum glucose levels by Zn lectin complex treated group is at lower dose than lectins for both oral and subcutaneous route of administration. 12
EFFECT OF LECTINS & ZINC FREEZE DRIED LECTINS COMPLEX ON SERUM TOTAL CHOLESTEROL LEVELS 160 Serum Cholesterol Levels mg/dl 140 Normal Control (No Alloxan) 120 Diabetic Control 100 Glibeclamide (Standard) 80 250mg/kg bw.s.c (lectins) 60 350mg/kg bw.p.o (lectins) 40 150mg/kg bw.s.c (Zn lectin 20 complex) 150mg/kg bw.p.o (Zn lectin 0 complex) Day 0 Day 1 Day 14 Treatment period Decrease in total serum cholesterol levels indicate that some of the serious symptoms related to high cholesterol levels can be controlled by both lectins & zinc lectins complex treatment. 13
EFFECT OF LECTINS & ZINC FREEZE DRIED LECTINS COMPLEX ON TOTAL SERUM TRIGLYCERIDES LEVELS 140 Serum triglyceride levels mg/dl 120 Normal Control (No Alloxan) 100 Diabetic Control Glibeclamide (Standard) 80 250mg/kg bw.s.c (lectins) 60 350mg/kg bw.p.o (lectins) 40 150mg/kg bw.s.c (Zn lectin 20 complex) 150mg/kg bw.p.o (Zn lectin 0 complex) Day 0 Day 1 Day 14 Treatment period The reduction in the elevated triglycerides levels was seen on treatment with lectins & Zn lectin complex. However, Zn lectins show the effect at much lower dose than lectins. 14
EFFECT OF LECTINS & ZINC FREEZE DRIED LECTINS COMPLEX ON LIVER GLYCOGEN CONTENT 40 35 Liver Glycogen Content mg/g 30 of wet tissue 25 20 15 10 5 0 Normal Ctrl Diabetic Ctrl Standard 250mg/kg 350mg/kg 150mg/kg 150mg/kg bw.p.o (Zn bw.s.c bw.p.o bw.s.c (Zn (lectins) (lectins) lectin) lectin) Glibenclamide, Lectins & Zn lectin complex show restoration of liver glycogen levels which suggests lectins may bind to allostearic sites of insulin thus promoting action of insulin. 15
EFFECT OF LECTINS & ZINC FREEZE DRIED LECTINS COMPLEX ON BODY WEIGHT 15 % Change in mean body wt (Gain/Loss) 10 5 Normal Control (No alloxan) Diabetic Control 0 Glibeclamide (Standard) Day 1 Day 7 Day 14 250mg/kg bw.s.c (lectins) -5 350mg/kg bw.p.o (lectins) 150mg/kg bw.s.c (Zn lectin complex) -10 150mg/kg bw.p.o (Zn lectin complex) -15 -20 Treatment Period The diabetic rats treated with lectins & Zn lectin complex by subcutaneous as well as oral route did not reveal weight gain respectively as compared to standard Gilbenclamide. 16
C ONCLUSION The Vigna radiata seed contains galactose specific lectin. The MBL-I may be a tetrameric protein with molecular weight 160-180 kDa and may be composed of identical or nearly identical relative subunits of molecular weight of 45-50 kDa. Binding of the lectins with zinc improves stability and efficacy of the lectins. The lectin obtained from Vigna radiata seeds and zinc lectin complex has a good potential to be explored as a safe natural antidiabetic agent acting with controlled reduction in blood glucose levels. 17
Future Scope Antidiabetic potential of zinc lectins complex can be further explored by Streptozotocin model. Insulin mimicking activity can be studied for the lectins using in vitro, in vivo models to establish mechanism for antidiabetic activity and cholesterol lowering effect. 18
References o Somta, C., Somta, P., Tomooka N., C-Ooi, P.A., Vaughan D.A. and Srinives, P., �Characterization of new sources of mungbean ( Vigna radiata (L.) Wilczek) resistance to bruchids, Callosobruchus spp. (Coleoptera: Bruchidae )�, Journal of Stored Products Research, 2008, 44, 316-321. o Goldstein, I.J. and Poretz, R.D., �Isolation, physicochemical characterization, and carbohydrate-binding specificity of lectins �, in The Lectins: Properties, Functions, and Applications in Biology and Medicine, Liener, I.E., Sharon, N. and Goldstein, I.J. (Eds), Academic Press. Inc, Orlando, Florida, 1986, 33-50. o Patent Filed: Antidiabetic Lectins from Vigna radiata and its complex with Zinc., Indian Patent Office August 25, 2010. (Patent Appl No. 2370/MUM/2010) 19
A NATURAL ANTIDIABETIC AGENT CAN BE ALWAYS HELPFUL IN PROPHYLAXIS PATIENTS 20
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