radiata as potential anti-diabetic agent Chhaya Gadgoli 1, *, Lalit - - PowerPoint PPT Presentation

Complex of zinc and lectins from seeds of Vigna radiata as potential anti-diabetic agent

Chhaya Gadgoli1,*, 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

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Complex of zinc and lectins from seeds of Vigna radiata as potential anti-diabetic agent

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Crude drug Macerati

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Centrifugation Precipitatio n Dialysis Gel Affinit y SDS- PAGE Western Blot

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Zn Lectin Complex Invivo study

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

• f 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

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Introduction

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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.

Results and Discussion

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CHARACTERIZATION OF LECTINS

HEAMAGGLUTINATION ACTIVITY: Performed by Standard dilution technique using rabbit erythrocyte suspension. The effect

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sugars, pH and temperature

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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.

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GEL ELECTROPHORESIS

SDS-PAGE data revealed the presence of a single sharp in the region of 45-50 kDa Native-PAGE data revealed the presence of a single sharp in 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.

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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.

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PREPARATION 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)

TEM & DSC Analysis of Lectins and Zinc Lectin Complex

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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 Avg Particle Size 1.259µm Avg Particle Size 1.326µm

Acute toxicity studies (as per OECD 425)

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IN-VIVO EVALUATION OF LECTINS & ZINC LECTIN COMPLEX Treatment group (Female Wistar rats) Dose Number of animals Mortality

• bserved

Saline suspension of lyophilized crude lectins 2000 mg/kg b.w.s.c. 06 No mortality 2000 mg/kg b.w.p.o. 06 No mortality Saline suspension of Zinc freeze dried lectins complex 2000 mg/kg b.w.s.c. 06 No mortality 2000 mg/kg b.w.p.o. 06 No mortality Saline suspension of crude lectins & Zn lectin complex is practically safe & its LD50 (MTD) is higher than 2000 mg/kg

ORAL GLUCOSE TOLERANCE TEST (OGTT)

The risk of hypoglycemia observed in Glibenclamide treated group is not

• bserved in lectins as well as Zn lectins complex treated group.

11 20 40 60 80 100 120 140 160 180 200 30 60 90 120 240

Blood Sugar Levels mg/dl Time in (Minutes)

Normal Control Glucose Control Glibeclamide (Standard) 150mg/kg bw.s.c (zn lectin complex) 250mg/kg bwp.o (zn lectin complex) 300 mg/kg bw s.c (lectins)

+Glucose

EFFECT OF FREEZE DRIED LECTINS & ZINC LECTIN COMPLEX ON SERUM GLUCOSE LEVELS

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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.

50 100 150 200 250 300 350 Day 0 Day 1 Day 7 Day 14

Serum Glucose Levels mg/dl Treatment Period (Days)

Normal Control Diabetic Control Glibeclamide (Standard) 250mg/kg bw.s.c (lectins) 350mg/kg bw.p.o (lectins) 150mg/kg bw.s.c (Zn lectin complex) 150mg/kg bw.p.o (Zn lectin complex)

EFFECT OF LECTINS & ZINC FREEZE DRIED LECTINS COMPLEX ON SERUM TOTAL CHOLESTEROL LEVELS

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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.

20 40 60 80 100 120 140 160 Day 0 Day 1 Day 14 Treatment period

Serum Cholesterol Levels mg/dl

Normal Control (No Alloxan) Diabetic Control Glibeclamide (Standard) 250mg/kg bw.s.c (lectins) 350mg/kg bw.p.o (lectins) 150mg/kg bw.s.c (Zn lectin complex) 150mg/kg bw.p.o (Zn lectin complex)

EFFECT OF LECTINS & ZINC FREEZE DRIED LECTINS COMPLEX ON TOTAL SERUM TRIGLYCERIDES LEVELS

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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.

20 40 60 80 100 120 140 Day 0 Day 1 Day 14 Treatment period

Serum triglyceride levels mg/dl

Normal Control (No Alloxan) Diabetic Control Glibeclamide (Standard) 250mg/kg bw.s.c (lectins) 350mg/kg bw.p.o (lectins) 150mg/kg bw.s.c (Zn lectin complex) 150mg/kg bw.p.o (Zn lectin complex)

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EFFECT OF LECTINS & ZINC FREEZE DRIED LECTINS COMPLEX ON LIVER GLYCOGEN CONTENT

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.

5 10 15 20 25 30 35 40

Normal Ctrl Diabetic Ctrl Standard 250mg/kg bw.s.c (lectins) 350mg/kg bw.p.o (lectins) 150mg/kg bw.s.c (Zn lectin) 150mg/kg bw.p.o (Zn lectin)

Liver Glycogen Content mg/g

• f wet tissue

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EFFECT OF LECTINS & ZINC FREEZE DRIED LECTINS COMPLEX ON BODY WEIGHT 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.

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• 15
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5 10 15 Day 1 Day 7 Day 14

% Change in mean body wt (Gain/Loss)

Treatment Period

Normal Control (No alloxan) Diabetic Control Glibeclamide (Standard) 250mg/kg bw.s.c (lectins) 350mg/kg bw.p.o (lectins) 150mg/kg bw.s.c (Zn lectin complex) 150mg/kg bw.p.o (Zn lectin complex)

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CONCLUSION

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.

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Future Scope

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Antidiabetic potential of zinc lectins complex can be further explored by Streptozotocin model.

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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.

References

• 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.

• 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.

• Patent Filed: Antidiabetic Lectins from Vigna radiata and its

complex with Zinc., Indian Patent Office August 25, 2010. (Patent Appl No. 2370/MUM/2010)

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