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Project Report

On Major Research Project

Ref. No. 41 – 1262/2012 (SR), 26 July 2012

“Studies on bioactive phytopeptides of Zizyphus species with

special reference to lectins and investigation in to their

Immunomodulatory action”

Principal Investigator

Dr. Ms. M. B. PATIL, Professor & Head

Post Graduate Teaching Department of Biochemistry

RTM Nagpur University, LIT Premises, Amravati Road,

Nagpur – 440 033. (Maharashtra)

Project Report

On

Major Research Project

“Studies on Bioactive Phytopeptides of Zizyphus

Species With Special Reference to Lectins and

Investigation in to their Immunomodulatory action”

Ref. No. 41 – 1262/2012 (SR), 26 July 2012

Principal Investigator

Dr. Ms. M. B. PATIL, Professor & Head

Post Graduate Teaching Department of Biochemistry

RTM Nagpur University, LIT Premises, Amravati Road,

Nagpur – 440 033. (Maharashtra)

OBJECTIVES OF THE PROJECT :

Four varieties of Zizyphus species available in the vidharbha region, viz., Z.

mauritiana, Z. oenoplia, Zizyphus jujube and Z. xylopyra were selected.

1. To screen all parts of the plant for the presence of lectins.

2. To isolate and purify them to purity by using all standard purification

protocols.

3. To characterize them and determine all the properties of purified protein.

4. To study appearance of lectins during growth and development of plants,

leaves and seeds.

5. To study antibacterial, antifungal, carbohydrate binding and

Immunomodulatory action ‘in vitro’ using the isolated cell lines and ‘in vitro’

using experimental animal.

Work done (Period) : 1 October 2012 to 30 Sept. 2015

Zizyphus varieties used : Four varieties of Zizyphus identified by

taxonomist of the region were used for the

research project.

1. Zizyphus mauritiana

2. Zizyphus oenoplia

3. Zizyphus jujuba

4. Zizyphus xylopyra

Parts of the plants used : Leaves, flower, fruit and seed cotyledons were

collected from the plants capable of

producing fruits in fruiting season.

Time of collection of sample : November to March (2013/ 2014/ 2015) in the

fruiting season.

Animals used for : 1. Rabbits (Blood) – For haemagglutination

assay

: 2. Wistar Albino Rats – For Immunomodulatory

activity.

SR.NO.

TITLES

PAGE

NO.

1 INTRODUCTION 1-3

2 ISOLATION, PURIFICATION AND CHARECTERIZATION 4-35

2.1 INTRODUCTION 4

2.2 OBJECTIVES 4

2.3 MATERIALS AND METHODS 5-10

2.4 Screening of all parts for the presence of letins (Objective : 1) 6

2.5 Isolation and purification of lectins (Objective : 2) 6

2.5.1 Extract Preparation 6

2.5.2 Ammonium Sulphate fractionation 7

2.5.3 Purification of lectins by Affinity Chromatography 7

2.5.4 Purification of lectins by Ion-Exchange Chromatography 7

2.5.5 Protein Estimation 7

2.5.6 Phenol Sulphuric acid test 7

2.6 Characterization and determination of all properties of lectins (Objective: 3 ) 8-

2.6.1 Polyacrylamide Gel Electrophoresis 8

2.6.2 Agglutination Assay / Blood Group Specificity 8

2.6.3 Agglutination inhibition assay 8

2.6.4 pH Stability 9

2.6.5 Effect of temperature and thermal inactivation 9

2.6.6 Effect of metal ions on agglutination activity 9

2.6.7 Effect of inhibitor on agglutination activity 9

2.6.8 α and β- galactosidase activity 10

2.6.9 To study appearance of lectins during growth and development (Objective: 4) 10

2.7 RESULTS 11-33

2.7.1 Screening all parts of the plant for the presence of lectins 11

2.7.2 Purification of lectins from Zizyphus species 11

2.7.3 Affinity chromatography on Guar-Gum of leaves and seed lectins of Zizyphus

species

13

2.7.4 Ion Exchange chromatography on DEAE-Cellulose for seed lectins of

Zizyphus species

13

2.7.5 SDS Polyacrylamide gel electrophoresis of purified lectins 18

2.7.6 Agglutination Assay / Blood Group Specificity 18

2.7.7 Agglutination inhibition assay 19

2.7.8 pH Stability 19

2.7.9 Effect of temperature and thermal inactivation 19

INDEX

2.7.10 Effect of metal ions on agglutination activity 19

2.7.11 Effect of inhibitor on agglutination activity 19

2.7.12 α and β- galactosidase activity 20

2.7.13 Appearance of lectins during growth and development of plants. 19

2.8 CONCLUSION 34-35

3 ANTIBACTERIAL, ANTIFUNGAL, CARBOHYDRATE BINDING

AND IMMUNOMODULATORY ACTIVITY OF LECTINS

(Objective : 5)

36-67

3.1 INTRODUCTION 36

3.2 MATERIALS AND METHODS: 37-43

3.2.1 Antibacterial activity of purified lectins of Zizyphus species 38

3.2.2 Antifungal activity of purified lectins of Zizyphus species 38

3.2.3 Carbohydrate binding activity of purified lectins of Zizyphus species 39

3.2.4 Methods for in vitro immunomodulatory assay 39

3.2.4.1 Splenocyte proliferation assay 39

3.2.4.2 Lymphocyte proliferation assay 40

3.2.4.3 Phagocytic index assay 40

3.2.4.4 Lysosomal enzyme activity assay 40

3.2.5 Methods for in vivo immunomodulatory assay 41

3.2.5.1 Acute toxicity study 41

3.2.5.2 Cytokine assay (TNF-α) and Nitric oxide (NO) Estimation 42

3.2.5.3 Hemagglutination Assay 42

3.2.5.4 Effect of lectins in preventing systemic anaphylactic shock 42

3.2.5.5 Arthus Reaction 43

3.3 RESULTS 44-64

3.3.1 Antibacterial activity of purified lectins of Zizyphus species 44

3.3.2 Antifungal activity of purified lectins of Zizyphus species 45

3.3.3 Carbohydrate binding activity of purified lectins of Zizyphus species 46

3.3.4 In vitro and in vivo immunomodulatory activity of purified lectins of

Zizyphus species

49

3.3.4.1 Cytotoxic activity by MTT assay 49

3.3.4.2 Splenocyte proliferation assay 50

3.3.4.3 Lymphocyte proliferation assay 50

3.3.4.4 Phagocytic index assay 50

3.3.4.5 Lysosomal enzyme activity assay 50

3.3.4.6 Cytokine assay (TNF-α) and Nitric oxide (NO) Estimation 59

3.3.4.7 Hemagglutination Assay 60

3.3.4.8 Effect of lectins in preventing systemic anaphylactic shock 60

3.3.4.9 Effect of lectins in preventing Arthus reaction 62

3.4 CONCLUSION 65-67

4 REFERENCES 68-75

ANNEXURE - I 77

Project Report, UGC MRP, Ref. No. 41 – 1262/2012 (SR), 1 October 2012 - 15

1

1. INTRODUCTION

The immune system is extremely multipurpose defense system that has evolved to

protect animals from invading pathogenic microorganisms and cancer. It is able to generate

an enormous variety of cells and molecules capable of specifically recognizing and

eliminating an apparently limitless variety of foreign invaders. These cells and molecules act

together in a dynamic network whose complexity rivals that of the nervous system (Kuby, et

al., 2007). Immune system is a system of biological structures and process within an

organism protecting against diseases (Cruse and Levis, 1995).

Lectins, which are most commonly found in all types of organisms, animal and

plants, are widespread natural products with striking biological activities. Their specific

ability to recognize and bind to simple or complex saccharide facilitates their role as effective

information protein molecules. Functioning as agent of cell to cell recognition, lectins

promote symbiosis between plants and specific nitrogen-fixing bacteria. As natural defensive

molecules, they can protect plants against predator bacteria, fungi and insects. As part of our

diet, lectins are powerful exogenous growth factor in the small intestine and influence our

health, the digestive function and bacterial ecology of the alimentary tract. Lectins are also

important research tool in preparative biochemistry and cell science. Lectins are complex and

heterogenous group of carbohydrate binding proteins with diverse molecular structures,

biochemical properties and carbohydrate binding specificities. In addition, plant lectins can

be used to study glycoconjugates (Licastro et al., 1993).

They are abundantly distributed in nature, particularly in the plant kingdom where

they are found in seeds, leaves (Cavada et al., 1998), bark (Witisuwannakul et al., 1998),

bulbs (Yagi et al., 2002), rhizomes, roots (Oliveira et al.; 2002), cotyledons and tubers

(Konozy et al., 2002 and 2003). Leguminous seeds contains huge amounts of lectins that are

similar to those present in other tissues of same plant, including the latex. Plant lectins are

defined to be plant proteins, possessing at least one non catalytic domain, which bind

reversibly to a specific mono or oligosaccharide (Van Damme et al.; 1998). Complex

oligosaccharide structures present on the cell surface, effect of movement of glycoconjugates

to the surface mediating cell to cell and cell to matrix recognition events (Dodd and

Drickamer, 2001). By virtue of their binding capabilities, lectins are thus involved in diverse

mechanism such as endocytosis, intracellular translocation of glycoproteins, cellular

regulation, migration and adhesion, phagocytosis and binding of microorganism to host cells

(Sharon and Lis, 1973). Lectin specificity is usually defined according to the mono – or

oligo-saccharide that is able to inhibit the agglutinating activity induced by the lectin.

Stillmark first discovered lectins in year 1888. He worked with the extract of caster

beans (Ricinus communis) (Stillmark, 1888). He reported that the preparation he obtained

able to agglutinate red blood cells. Exactly one decade after the discovery of lectins,


2

Elfstrand, (1898) proposed the term hemagglutinin because hemagglutinating substances

were identified from other plants also. Hemagglutinins like ricin, abrins were found to be

highly toxic and due to this toxicity, it was relatively easy to test on animals. Indeed these

reports, the blood group specificity have led Boyd to propose the term lectin that was

derived from the word legree (in latin it means to select) and is referred to as the birth of

contemporary lectinology (Boyd and Shapleigh, 1954). A landmark in the discovery was

observed when Morgan and Watkins, (1953) laid the foundation of knowledge of the

environment of strict sugar specificity of agglutination reaction within human A+, B+ and O+

blood group system. Indeed the first proper definition of lectin was based on the sugar

specificity of the inhibition of hemagglutination reaction. Accordingly lectins are multivalent

binding proteins of non- immune origin showing agglutination with cells or precipitates

glycoproteins or polysaccharides (Goldstain et al.,1980).

Zizyphus is one of the plant commonly used in Egyptian and Chinese folk medicine

for treatment of various diseases. The leaves of the Zizyphus mauritiana are reported to have

hypoglycemic activity (Morton, 1987) as well as hyperglycemic activity and seed show

sedative effect (ISSG database, 2009). Beside this Z. mauritiana is an important food crop.

The fruits are rich in vitamins A, B, C, sugars, rare minerals and proteins. The leaves provide

fodder to cattle. In sore throat a leaf decoction is used as a gargle, for diarrhea and dysentery

decoction of root and stem bark is prescribed. The leaves are chewed in bleeding gums and in

treatment of mouth ulcers. The seed kernels are considered as aphrodisiac (Agroforestree,

2009).

Ziziphus oenoplia M., belonging to family Rhamnaceae (vernacular name: Siakul) is

a shrub, distributed in tropical and subtropical India in dry climates. The roots are astringent,

bitter, antihelmintic, digestive, and antiseptic. They are useful for treating hyperacidity,

ascaris infection, abdominal pain, and healing of wounds (NISC, 1998). The angiogenic

potential was also shown by of Ziziphus oenoplia root ethanolic extract using the

chorioallantoic membrane model (Mahapatra et al., 2011).

Zizyphus jujuba commonly called, Red date, Chinese date or Bera (Pushto), belongs

to family Rhamnaceae. This family consists of 50 genera and more than 900 species; it is

almost cosmopolitan and found mainly in subtropical to tropical areas. The bark, leaves and

fruit of several species of Rhamnaceae have been used as laxatives, notably Rhamnaceae

cathartica and Rhamnaceae frangula. Many Ziziphus species yield edible fruit, among these

are: Z. jujuba (Chinese jujube) and Ziziphus mauritiana (Indian jujube) which are cultivated

on a commercial scale. The roots of Ziziphus oxyphylla Edgew and juice of fresh leaves of Z.

mauritiana L are used for curing jaundice (Gul et al., 2009). A cold suspension of dried roots

and powder of Ampelozyziphus amazonicus is used to prevent malaria (Neto et al., 2008).

Extracts of Zizyphus jujuba are well studied and proven to be antibacterial, phytotoxic and

hemagglutination activities (Ahmad et al., 2011).


3

The stem bark decoction of Z. numularia is taken thrice a day for six days in diarrhea.

The leaves of Z. xylopyrus are chewed thrice a day for fifteen days in urinary troubles. The

Zizyphus plants have been shown to contain many saponins, glycosides, derivative of

glycosides, essential oils, used in the preparation of many pharmasuitical preparation, for

treatment of cancer, neurological disorders, antihistamine agents, antidepresents (Schomburg

et al., 2001)

Taking into consideration the properties of lectins and the properties of genus

Zizyphus, the plant were found to contain lectins (Gupta and Srivastava, 1998). Many wild

medicinal plants possess significant immunomodulatory potential can be used to endorse

positive health and maintain the resistance to infections. A wide variety of compounds of

potential medicinal value is found in the plant kingdom and featured in traditional cures.

Approximately half of the drugs in current use throughout the world still originate from

plants.

Many low molecular weight compounds like lectins, alkaloids, flevonoids, phenols,

saponins and terpenoids have been reported to have Immunostimulatory activity. The seeds

of Zizyphus species are reported to have immunostimulatory activity but scanty of work is

available another parts. Also no systematic approach has been initiated to study the various

modes of action of compounds isolated from Zizyphus species as far as immunomodulation is

concernd.

This work was done to investigate characterization of the lectins isolated from

Zizyphus species for antibacterial antifungal, carbohydrate binding and Immunostimulatory

action in vitro using isolated cell lines and in vivo using the experimental animals for

Immunostimulatory activity along with its mechanism of action.


4

2. ISOLATION, PURIFICATION AND CHARECTERIZATION OF

LECTINS

2.1. INTRODUCTION

Lectins are the protein or glycol-protein that have binding capacity selectively and

without the participation of enzymes, specific for carbohydrate ligands (Vijayan and chandra,

1999, Imberty et al., 2005). Lectins, a class of sugar-binding and cell agglutinating proteins

are ubiquitous in nature, being found in all kinds of organisms, from viruses to humans

(Sharon, 2008).

Advances in plant lectin biochemistry have made great strides during the past decade.

Technical advances in biophysical techniques and molecular biology, the availability of

synthetic oligosaccharides and characterization of lectins with unique carbohydrate binding

properties are responsible for these advances (Singh et al., 1999 ; Xu et al., 2007).

In the present report the data on investigations undertaken to purify lectins from

different parts of four Zizyphus species namely Zizyphus mauritiana, Zizyphus oenoplia,

Zizyphus jujuba and Zizyphus xylopyra has been reported. The purified lectins were

characterized by using all conventional and standard protocols.

2.2. OBJECTIVES:

1. To screen all parts of the plant for the presence of lectins.

2. To isolate and purify them to purity by using all standard purification protocols.

3. To characterize them and determine all the properties of purified lectin.

4. To study appearance of lectins during growth and development of plants, leaves

and seeds.


5

2.3. MATERIALS AND METHODS:

2.3.1 Plant material:

Leaves, flowers, fruits, of Zizyphus species were collected from Department of

Biochemistry of the RTM Nagpur University and were authenticated in the Herbarium of the

Department of Botany of the RTM Nagpur University, Nagpur, India. Plant Leaves were

dried under shade and were ground using grinder. The dried powder was then stored in

vacuum jar and used for extraction. Fruits were collected in fruiting season from single

growing plants and seeds were dried in sunlight, cotyledons were removed at the time of

experiment.

Figure. 2.1. Herbarium of authentication of Zizyphus species

Zizyphus mauritiana, Voucher No.: 9351 Zizyphus oenoplia, Voucher No.:9759

Zizyphus jujuba, Voucher No.: 9847 Zizyphus xylopyra, Voucher No.: 9848


6

2.3.2. Blood Samples:

Human blood samples of groups A+, B+ and O+ were collected from the healthy donor

from the Clinical Biochemistry Laboratory, University Department of Biochemistry, RTM

Nagpur University, Nagpur (MS), India.

Rabbit blood samples were collected from the departmental Animal and Breeding

House, University Department of Biochemistry; RTM Nagpur University, Nagpur (MS),

India.

2.3.3. Chemicals:

Bovine serum albumin, Guar gum, DEAE-Cellulose, Sephadex G-75, D-Arrabinose,

D-ribose, D-Xylose, α-D-Glucose, D-fructose, D-Mannose, D-Maltose, D-Galactose, D-

Mannitol, Sucrose, Lactose, Raffinose, N-Acetyl D-Glucosamine, N-Acetyl D-

galactosamine, Acrylamide, N-N-Methylene bisacrylamide, ammonium persulphate,

Tetramethylendiamine, Glycine, Tris-hydroxy-methyl aminomethylene diamine,

Bromophenol blue, Glycerol, Coomassie brilliant blue, Glacial acetic acid, Methanol,

Sodium carbonate, Copper sulphate, were obtained from Sigma chemicals, St Louis M.

O.,USA. Low molecular weight protein marker purchased from Genei, Bangalore, and all

other reagents were of analytical grade.

2.4. SCREENING OF ALL PARTS FOR THE PRESENCE OF LECTIN: (Objective: 1)

The leaves, flowers, fruit, seed cotyledons were collected and extracted with 0.1 – 1

M NaCl, PBS and deionised water for 2 h at 4oC. The suspensions were filtered through

cheese cloth and then centrifuged at 9668 g for 30 min at 4oC. The supernatants were

dialyzed against the extraction medium. The dialysed extracts were used for identification of

lectin activity.

2.5. ISOLATION AND PURIFICATION OF LECTINS: (Objective: 2)

Purification Procedures:

2.5.1. Extract preparation (10%):

The leaf powder of the four Zizyphus species (mentioned in materials) crushed and

extracted with phosphate buffer 0.02 M, pH-7. The suspension were filtered through cheese

cloth and then centrifuged at 9668 g for 10 min at 4oC (Remi C-24). The supernatants were

collected and termed as crude extract of leaves.

Seed cotyledons were crushed in the 0.5 M NaCl and processed as described for

leaves and termed as crude extract of seed.


7

2.5.2. Ammonium Sulphate fractionation:

To the crude extract were fractionated by ammonium sulphate (0 – 20, 20 – 60, 60 –

90%). The precipitates were suspended in minimum volume of extraction solution and the

suspensions were dialyzed against the same in the 4oC for 14 h with two changes of solution.

The dialyzed solution from each fraction (0 – 20, 20 – 60, 60 – 90%) which showed

agglutination was designated as ammonium sulphate fractions (Dixon, (1953). The

ammonium sulphate fractions, which showed agglutination were further purified by affinity

chromatography on cross-linked guar gum (Appukuttan et al., 1977).

2.5.3. Purification of lectin by affinity chromatography:

Cross linked guar gum column was prepared by the method of Appukuttan et al .,

(1977). Active ammonium sulphate fractions were loaded on the column and washed with

o.1M galactose till all proteins were removed. Active affinity chromatography fractions were

further fractionated by ion exchange chromatography on DEAE cellulose column.

2.5.4. Purification of lectins on Ion-Exchange chromatography:

Active affinity chromatography fractions were loaded on previously activated DEAE

cellulose column. Column elution was done by stepwise gradient of sodium chloride from

0.05M to 0.25M in sodium phosphate buffer (0.02 M, pH - 7) (Ali at al., 2012; Nisha et

al.,2013).

2.5.5. Protein estimation:

Protein concentration of of ZML-L, ZOL-L, ZJL-L, ZXL-L, ZMS-L, ZOS-L, ZJS-L, ZXS-L

was determined by the method of Lowry et al., (1951).

2.5.6. Phenol sulphuric acid test:

Carbohydrate estimation was performed by phenol sulphuric acid test using D-glucose as

standard (Dubois et al.,1956).

The purified lectins were designated as ZML-L (Purified lectin from leaves of

Zizyphus mauritiana), ZOL-L (Purified lectin from leaves of Zizyphus oenoplia), ZJL-L

(Purified lectin from leaves of Zizyphus mauritiana jujuba), ZXL-L (Purified lectin from

leaves of Zizyphus xylopyra), ZMS-L (Purified lectin from seeds of Zizyphus mauritiana),

ZOS-L (Purified lectin from seeds of Zizyphus oenoplia), ZJS-L (Purified lectin from seeds

of Zizyphus jujuba), ZXS-L (Purified lectin from seeds of Zizyphus xylopyra).


8

2.6. CHARECTERIZATION AND DETERMINATION OF PROPERTIES OF

LECTIN: (Objective: 3)

2.6.1. Polyacrylamide Gel Electrophoresis (PAGE):

Homogeniety of lectins from Zizyphus species (ZML-L, ZOL-L, ZJL-L, ZXL-L,

ZMS-L, ZOS-L, ZJS-L, ZXS-L) was determined by the method described by Weber and

Obsborn, (1969). Acrylamide concentration of separator gel was 10% and that of stacking gel

was 5% (Weber and Obsborn, 1969; Davis, 1964).

Table 2.1. Preparation of gel for polyacrylamide gel electrophoresis

Reagents and chemicals Running gel composition

(5%)

Stacking gel composition

(5%)

Distilled water 3.18ml 6.3ml

Acrylamide monomer 9.01ml 1.5ml

Separating buffer 5.2ml --

Stacking gel buffer -- 1.2ml

SDS (10%) 0.2ml 0.09ml

Ammonium per sulfate 0.1ml 0.09ml

TEMED 0.01ml 0.03ml

2.6.2. Agglutination Assay / Blood Group Specificity:

Preparation of erythrocyte suspension:

Human blood group A+, B+, O+ and rabbit erythrocytes were prepared and used by the

method of Dhingra et al., 1995.

Hemagglutination Assay / blood group specificity:

Human blood group A+, B+, O+ and rabbit erythrocytes suspensions were used to

determination of hemagglutination assay by the method of Suseelan et al., 1997.

2.6.3. Agglutination inhibition assay:

Haemagglutination inhibition assay were performed by mixing 50 µl of ZML-L, ZOL-L,

ZJL-L, ZXL-L, ZMS-L, ZOS-L, ZJS-L, ZXS-L with an equal volume of 0.1 M different

carbohydrates (D-Arrabinose, α-D-Glucose, D-fructose, D-Mannose, D-Maltose, D-

Galactose, D-Mannitol, Sucrose, Lactose, Raffinose, p-nitrophenyl-α-D-galactopyranoside,

o-nitrophenyl-β-D-galactopyranoside), using the method suggested by Kurokawa et al.,

(1976).


9

2.6.4. pH Stability:

The buffers used to study the stability of ZML-L, ZOL-L, ZJL-L, ZXL-L, ZMS-L,

ZOS-L, ZJS-L, ZXS-L under different condition of pH 1 to 13 were as follows, for pH 1 –

0.01 N HCl, for pH 2 and 3 – 0.2 M glycine – HCL buffer, for pH 4 and 5, 0.2 M sodium

acetate buffer, for pH 6 and 7 – 0.2 M sodium phosphate buffer, for pH 8 – 0.2 M Tris –HCL

buffer, for pH 9 – 0.2 M glycine – NaOH buffer, for pH 10, 11, 12 and 13 – 0.2 M carbonate

– bicarbonate buffer. One hundred µl lectins solutions and one hundred µl of buffer solutions

were incubated for 1 h at ambient temperature. Aliquots were withdrawn and assayed for

agglutination as mentioned previously (Suseelan et al., 1997).

2.6.5. Effect of Temperature and Thermal Inactivation:

The heat stability of hemagglutinating activity of of ZML-L, ZOL-L, ZJL-L, ZXL-L,

ZMS-L, ZOS-L, ZJS-L, ZXS-L was determined by incubation of aliquots of lectins solutions

at different temperatures. To examine the thermostability, thirty µl of lectins solution was

added to one ml 0.006 M sodium phosphate buffer pH 7 and were incubated for various

periods at 20, 40, 60, 80, and 100 oC for 1 h. Aliquots were withdrawn and assayed for

agglutination at ambient temperature after cooling as mentioned previously (Suseelan et al.,

1997).

2.6.6. Effect of metal ions on agglutination activity:

The metal ion requirement for ZML-L, ZOL-L, ZJL-L, ZXL-L, ZMS-L, ZOS-L, ZJS-

L and ZXS-L activity was examined by demetalizing the samples and then treating with

different metal ions. Lectins solutions (100 µg of 4.5µg/µl) were taken in an eppendorf tube

and incubated with four hundred µl of 10 mM EDTA at pH 5.0 for 20 h at 4oC. The samples

was then dialysed against 25 mM citrate buffer pH 5 and 50 µl aliquots were transferred to

eppendorf tubes containing fifty µl of 1 mM CaCl2, MnCl2, MgCl2, HgCl2, ZnCl2, FeCl2,

ZnSO4, PbCl2, 1,10-phenanthrolene and incubated for 2 h. Activity of samples were then

examined by agglutination assay as described (Kawagishi et al.,1990).

2.6.7. Effect of inhibitor on agglutination activity:

The effect of inhibitor on the agglutination activity of purified lectins i.e. ZML-L,

ZOL-L, ZJL-L, ZXL-L, ZMS-L, ZOS-L, ZJS-L and ZXS-L was determined by the method

given by the Deshpande and Patil (2003). The purified fractions were mixed with 1 mM

inhibitors solutions namely 1, 10-phenanthrolene, reduced glutathione, meso-inositol, SDS,

cystein hydrochloride, phenyl methyl sulphonylfluoride, 2-mercaptoethenol, para –

chloromercuric benzoate, n-bromosuccinamide and EDTA, and incubated at 37 oC for 1 h

and tested for hemagglutination with rabbit erythrocytes. Each of the purified lectins solution

without treatment with any inhibitor was used as a positive control and normal saline with

erythrocytes was used as a negative control.


10

2.6.8. α and β- galactosidase activity:

The substrates used in the study were α – p NPG (3 mM) and β – oNPG (3 mM). α and β-

galactosidase assays were carried out by the method of Murrey, (1983).

2.6.9. TO STUDY APPERANCE OF LECTINS DURING GROWTH AND

DEVELOPMENT: (Objective: 4)

Seedling of Zizyphus species were grown in the standard laboratory condition with an

adequate sunlight in departmental laboratory. The seeds were sown in sterile sand and soil

and the plants were grown under normal environmental condition in the month of July. The

plants were watered daily as and when required at regular interval by using sterile distilled

water (Pueppke, 1979).

The vegetative growth of the plants of Zizyphus species was observed to check the

optimum age of the plant for maximum lectin content. The whole plant from the each species

was uprooted at an interval of week up to 45 days after germination to check the existence of

lectin activity (Howerd et al.,1972).

The uprooted plants were extracted in 1M NaCl as described previously and were

subjected to hemagglutination assay and protein measurement as described.


11

2.7. RESULTS:

2.7.1. Screening all parts of the plant for the presence of lectins:

The leaves, flowers, fruit, seed cotyledons of Zizyphus species having more amounts

of lectin and protein concentration tested by hemagglutination assay (Suseelan et al., 1997)

and protein estimation (Lowry et al., 1951). Therefore the samples of leaves and cotyledons

were used for the purification (Table 2.2) and characterization of lectins.

Table 2.2. Screening of all parts of Zizyphus species for presence of lectin.

Sr.

No.

Zizyphus Species Parts of Plant Protein

Conc./ml

HAU/ml

1

Zizyphus mauritiana

Leaves 4.85 5120

Flowers 5.6 --

Fruit 2.3 80

Seed Cotyledons 15.4 20480

2

Zizyphus oenoplia

Leaves 7.6 5120

Flowers 5.2 --

Fruit 2.8 40


3

Zizyphus jujuba

Leaves 3.65 5120

Flowers 4.8 --

Fruit 2.4 40


4

Zizyphus xylopyra

Leaves 3.6 420

Flowers 4.3 --

Fruit 2.5 40

Seed Cotyledons 8 640

2.7.2. Purification of lectins from Zizyphus species:

The 20 – 60% ammonium sulphate precipitate was applied to cross linked guar-gum

column at 4oC, which was previously equilibrated with 0.02 M phosphate buffer pH 7.2.

Lectins were eluted as 3 major peaks i.e. F-1, F-2, and F-3. The fractions (F-1, F-2, F-3) were

pooled together and subjected to 100% ammonium sulphate precipitation (ASP) and

homogeneity was checked by polyacrylamide disc gel electrophoresis (Figure, 2.14). It was

evident from the electrophoresis pattern that some of the affinity chromatography fractions

(ACF) were not homogeneous proteins; therefore these ACF were loaded on the DEAE-

Cellulose column and elution was made by the concentration gradient of NaCl. The eluted

fractions which show hemagglutination activity were again checked for the homogeneity. It

was found that the ion exchange chromatography fractions (IEF) showed the homogeneity on

polyacrylamide gel electrophoresis and appeared as single band with repeated testing.


12

Table 2.3. Purification chart of Zizyphus mauritiana leaf lectin (ZML-L),

haemagglutination assays were conducted with a suspension of rabbit erythrocytes.

Purification step Volume

(ml)

Proteins

(mg/ml)

HAUa

SAb

Purification

fold

Yield %

Crude extract 100 4.85 5120 1055.6 1 100

ASP Fraction 25 1.9 10240 5389 5 50

ACF fraction 11 0.2 8800 44000 41.68 18.9

Table 2.4. Purification chart of Zizyphus oenoplia leaf lectin (ZOL-L).

Table 2.5. Purification chart of Zizyphus jujuba leaf lectin (ZJL-L).

Table 2.6. Purification chart of Zizyphus xylopyra leaf lectin (ZXL-L).


(ml) Protein conc.

(mg/ml) HAUa

SAb

Purification

fold

Yield

%

Crude extract 100 3.6 420 116 1 100

ASP Fraction 30 4.3 780 204 1.75 56.86

ACF Fraction 8 1.03 860 834 7.2 16.13

Purification

step

Volume

(ml)

Proteins

(mg/ml)

HAUa

SAb

Purification

fold

Yield %

Crude extract 100 7.6 5120 673.68 1 100

ASP Fraction 27 1.14 1280 1122.8 1.66 67.5

ACF fraction 10 0.04 800 20000 29.71 16


(ml)

Proteins

(mg/ml)

HAUa

SAb

Purification

fold

Yield %

Crude extract 100 3.65 5120 1402 1 100

ASP Fraction 13 0.5 10000 20000 14.26 25.39

ACF Fraction 7 0.03 8000 2666.6 19.02 10.93


13

Table 2.7. Purification chart of Zizyphus mauritiana seed lectin (ZMS-L).


(ml)

Proteins

(mg/ml)

HAUa

SAb

Purification

fold

Yield %

Crude extract 100 15.4 20480 1329.8 1 100

ASP Fraction 23 5.76 20480 3555.5 2.675 23

ACF Fraction 8 0.9 40960 45511 34.24 16

IEC Fraction 6 0.085 20480 240941 181.29 6

Table 2.8. Purification chart of Zizyphus oenoplia seed lectin (ZOS-L).


(ml)

Proteins

(mg/ml)

HAUa

SAb

Purification

fold

Yield %

Crude extract 100 9.5 2560 269.47 1 100

ASP Fraction 22 6.0 20480 853.33 3.1 80

ACF Fraction 16 3.84 10240 2666.66 9.91 64

IEC Fraction 10 1.10 5120 18618.18 69.21 44

Table 2.9. Purification chart of Zizyphus jujuba seed lectin (ZJS-L).


(ml)

Proteins

(mg/ml)

HAUa

SAb

Purification Yield %

Crude extract 100 8.65 2560 259.9 1 100

ASP Fraction 15 9 5120 568.8 2.18 8.9

ACF Fraction 10 3.2 10240 3200 12.31 40

IEC Fraction 7 1.2 20480 17066 65.66 56

Table 2.10. Purification chart of Zizyphus xylopyra seed lectin (ZJS-L).


(ml)

Proteins

(mg/ml)

HAUa

SAb

Purification

fold

Yield %

Crude extract 100 8 640 80 1 100

ASP Fraction 27 9 2560 284.44 3.55 85.7

ACF Fraction 15 3.6 5120 1422.2 17.77 66.7

IEC Fraction 8 1.03 5120 9941.7 124.2 64

For Table 2.2 – 2.9, HAU- Hemagglutination Unit, b) SA- Specific Activity


14

2.7.3. Affinity chromatography on Guar-Gum of lectins of Zizyphus species:

Elution profile of lectins from cross-linked guar gum column, 20-60% ammonium

sulphate fraction was loaded on the cross linked guar gum column equilibrate with 0.02 M

sodium phosphate buffer pH 7.2. The column was 14 x 2.5 cm (68ml bed volume). Fractions

of 5 ml each were collected and absorbance monitored at 280 nm (Figure,2.2 – 2.9). Lectins

are eluted as three major peak i.e. Fraction F-1, F-2, F-3 were pooled and precipitated 100%

ammonium sulphate saturation and dialysed against the extraction solution and the fractions

possessed hemagglutination activity. The ACF of leaf sample gave single band but seed

samples gave multiple bands on polyacrylamide gel electrophoresis, therefore, processed

them for ion exchange chromatography on DEAE-Cellulose.

2.7.4. Ion Exchange chromatography on DEAE-Cellulose for lectins of Zizyphus species:

Figure 2.10 – 2.13 shows DEAE-Cellulose ion exchange chromatography of

ammonium sulphate fraction (ASF) of seed lectins which gaves multiple bands on affinity

chromatography. The column was packed with the activated DEAE- Cellulose and ASF of

seed loaded on it the elution was made with the concentration gradient of NaCl, on the 0.15

M – 0.20 M concentration the absorbance at 280 nm gave single major peak. The fraction

further possessed hemagglutination activity and gave single band on polyacrylamide gel

electrophoresis.


Elution profile of Affinity chromatography of leaves and seed lectins of Zizyphus species


Elution profile of the Ion Exchange chromatography on DEAE-Cellulose for seed lectins of Zizyphus species


18

2.7.5. SDS Polyacrylamide gel electrophoresis of purified lectins

In SDS- polyacrylamide disc gel electrophoresis, leaf and seed lectins (ZML-L, ZOL-L, ZJL-

L, ZXL-L, ZMS-L, ZOS-L, ZJS-L and ZXS-L )of Zizyphus species show single band.

Standard molecular weight markers were run in side wells (Genei, Banglore). In Figure, 2.14

the SDS-PAGE electrophoretic pattern of Zizyphus lectins reveals a single band in lane 1,

corresponding to the molecular weight marker in lane 2.

Figure 2.14 SDS Polyacrylamide gel electrophoresis of purified lectins

ZMS-L: 27 Kd, ZOS-L: 25,kD ZJS-L: 29 kD ZXS-L: 23 kD

ZML-L: 23 Kd ZOL-L: 23Kd ZJL-L: 20 kD ZXl-L: 17 kD

Characterization and study all properties of purified lectins.

2.7.6. Agglutination Assay and Blood Group Specificity:

The purified fractions ZML-L, ZOL-L, ZJL-L, ZXL-L, ZMS-L, ZOS-L, ZJS-L and

ZXS-L had major binding capacity with rabbit and human B+ erythrocytes as compared to

human A+ and O+ erythrocytes (Figure, 2.15).


19

2.7.7. Hemagglutination Inhibition assay:

ZML-L, ZOL-L, ZJL-L, ZXL-L, ZMS-L, ZOS-L, ZJS-L and ZXS-L agglutinated

human as well as rabbit erythrocyte as shows in figure, 2.16. It was found from the results

that when rabbit erythrocytes incubated with lectins, D-galactose, para- nitrophenyl-α- D-

galactopyranoside, ortho- nitrophenyl-β-D-galactopyranoside, lactose and D-raffinose are the

most potent inhibitor for the Zizyphus seed and leaf lectins ( Figure, 2.16 – 2.23). These

results indicated that the purified lectins were galactose- binding lectins.

2.7.8. pH Stability:

The pH dependence of ZML-L, ZOL-L, ZJL-L, ZXL-L, ZMS-L, ZOS-L, ZJS-L and

ZXS-L was determined by incubating in buffers of varying pH. The optimum pH for

agglutination activity was found to be pH 7. All the purified lectins lost 50% activity between

pH 9 and 10 and were completely inactive at pH 2, 3, 11, 12 and 13 (Figure, 2.24 – 2.31).

2.7.9. Thermostability and thermal inactivation:

When ZML-L, ZOL-L, ZJL-L, ZXL-L, ZMS-L, ZOS-L, ZJS-L and ZXS-L were

heated from room temperature to 80oC at interval of 20oC, a linear relationship was observed

for percent residual activity vs. temperature. Purified lectins when heated at several

temperature ranging from 20 – 80oC for 1 h showed 100% activity indicating the lectins to be

thermostable. The lectins did not lose its agglutination activity when incubated at an ambient

temperature from 20 – 140 min (Figure, 2.32 – 2.39).

2.7.10. Effect of metal ions (Activators / Inhibitors) on agglutination activity:

Results in the figures 2.40 – 2.47 represent the effect of metal ions on

hemagglutination activity by ZML-L, ZOL-L, ZJL-L, ZXL-L, ZMS-L, ZOS-L, ZJS-L and

ZXS-L. It is found that heavy metal ions like Hg++, Sb++, Pb++ inhibit the activity of lectins.

While Mg++ and Mn++ completely restored the activity of purified lectin as compared to other

metal ions and controls.

2.7.11. Effect of Inhibitors on agglutination activity:

Treatment of purified lectins with different inhibitors solution such as SDS and 2-

mercaptoethenol gave complete inhibition of agglutinating activity as compared to other

inhibitors. Figure 2.48 – 2.55 shows that inhibitors used in this experiment inhibit the activity

of the purified lectins.


20

ENZYMATIC ACTIVITY

2.7.12. α-and β- galactosidase activity:

ZML-L, ZOL-L, ZJL-L, ZXL-L, ZMS-L, ZOS-L, ZJS-L and ZXS-L exhibited α-and

β-galactosidase activities with substrates α-PNPGal and β-ONPGal, respectively (Table

2.11). From the results it was found that the purified proteins have major β- activity as

compared to α.

Table 2.11. α-and β- galactosidase activity of purified lectins of seeds and leaves of

Zizyphus species.

Enzyme activities

U/ml ZML-L ZOL-L ZJL-L ZXL-L ZMS-L ZOS-L ZJS-L ZXS-L

α- galactosidase 11 13 8 10 13 15 10 12

β-galactosidase 26 27 22 19 29 32 26 22

2.7.13. Estimation of appearance of lectins during growth and development of plants.

Various parts of the plants of Zizyphus species was screened for the existence of

lectin activity. Figure 2.56 – 2.59 and figure 2.60 summarizes the result of the

hemagglutination assays of Zizyphus during germination and seasonal variation of lectin

content in photosynthetic leaves respectively.

The lectin level in the growing plants of Zizyphus species showed gradual increase in

lectin content initial days of growth 7 to 15 days after germination (Figure, 2.56 – 2.59)

during this period the plants were growing rapidly. The lectin content did not increase

continuously after 45 days of growth.

The seasonal variation of the lectin content in the leaves of Zizyphus species was estimated

round the year with the interval of 15 days. It was observed that in the flowering and fruiting

season the lectin content in the leaves was highest as compared to the non flowering &

fruiting season (Figure, 2.60).


Figure 2.15 Blood Group Specificity of leaf and seed lectins with human and rabbit erythrocytes:


35

2.8. CONCLUSION:

ZML-L, ZOL-L, ZJL-L, ZXL-L, ZMS-L, ZOS-L, ZJS-L and ZXS-L show maximum

agglutination with rabbit and human B+ erythrocytes. The agglutination was prevented by D-

galactose indicating these lectins to be galactose specific. The existence of a sugar binding

domain in these lectins was confirmed by affinity chromatography on cross linked guar gum,

the purified lectins were found to be monomeric in nature, with molecular weight of ZML-L:

23 kD, ZOL-L: 23 kD, ZJL-L: 20 kD, ZXL-L:17 kD, ZMS-L: 27 kD, ZOS-L: 25 kD, ZJS-L:

29 kD and ZXS-L: 23 kD.

The results shown in figure 2.15 for purified lectins showed the highest activity with

human B+ and rabbit erythrocytes from that ZOL-L, ZMS-L and ZOS-L showed highest

agglutinating activity as compared to ZML-L, , ZJL-L, ZXL-L, ZJS-L and ZXS-L.

ZML-L, ZOL-L, ZJL-L and ZXL-L were adsorbed by cross-linked guar-gum at pH 7

and lectins where purified by affinity chromatography. ZMS-L, ZOS-L, ZJS-L and ZXS-L

were also adsorbed on cross linked guar gum but cannot be purified by ion-exchange

chromatography on DEAE-Cellulose. A novel lectin having molecular weight 32.2 kD from

the wild mushroom Xerocomus spadicus was isolated by the same procedure of ion exchange

chromatography on DEAE- Cellulose (Quinhong et al., 2004).The abrin which is a toxic

protein having molecular weight 52 kD was also purified and characterized after isolation on

DEAE-Cellulose (Sjur Olsenes and Alekzander Pihl, 1973).

The agglutination activity of ZML-L, ZOL-L, ZJL-L, ZXL-L, ZMS-L, ZOS-L, ZJS-L

and ZXS-L were inhibited in the presence of galactose and galactose derivatives. It is evident

from the result of that galactose, para-nitrophenyl-α-D-galactopyranoside and ortho-

nitrophenyl-β-D-galactopyranoside, lactose and raffinose were able to inhibit agglutination.

Vigna radiate and Vigna mungo were found to be galactose specific (Hankins et al., 1980;

Hankins and Shennon, 1978).

Figure 2.24 – 2.31 shows a peak at pH 7, illustrating the activity of purified lectins is

stable at neutral pH. The agglutination activity was found to be stable between pH 4 to 10.

ZML-L, ZOL-L, ZJL-L, ZXL-L, ZMS-L, ZOS-L, ZJS-L and ZXS-L, when heated at

80oC for 1 h and exposed at 37oC from 20 to 100 min could not lose its agglutination activity

indicating the lectins to be thermostable (Figure 2.32 – 2.39).

It was also reported that all lectins required metal ions for agglutination (Galbrath and

Goldstein, 1972). ZML-L, ZOL-L, ZJL-L, ZXL-L, ZMS-L, ZOS-L, ZJS-L and ZXS-L

required metal ions like Mg++ and Mn++ to complete restore the activity of lectin, while


36

Zn++, Ca++, Fe++ also increased the agglutination activity as compared to demetalised (by

EDTA) lectin sample. The heavy metal ions like Hg++, Pb++ and Sb++ can complete inhibit

the agglutination activity (Figure 2.40 – 2.47).

ZML-L, ZOL-L, ZJL-L, ZXL-L, ZMS-L, ZOS-L, ZJS-L and ZXS-L exhibited both

α- and β- galactosidase activity with α- PNPGal and β-ONPGal.

Lectins have been purified from the crude extract of leaves and seeds of the

Zizyphus. All lectins are glycoproteins in nature as they gave positive test with phenol

sulphuric acid test.


37

3. ANTIBACTERIAL, ANTIFUNGAL, CARBOHYDRATE BINDING AND

IMMUNOMODULATORY ACTIVITY OF LECTINS

3.1. INTRODUCTION:

It has been reported that zizyphus is one of the richest genus of with phytochemicals

(Dimitris et al., 1997). Ethenolic extract of leaves and endocarp of Zizyphus mauritiana helps

to mentain level of glutathione, superoxide dismutase, catalase, vitamin E and decrease in

lipid peroxidation levels(Dhiru, 2005; Kalidose and Krishnamurthy,2011) indicating its

antioxidant property.

Zizyphus mauritiana has been reported for antimicrobial activity against Aspergillus

niger, Candida albicans, Enterobacter species, Escherichea coli, Klebsiella pneumonia,

Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus

species (Abalak et al., 2010 and Raghvendra et al., 2011). Plants rich in phytomolecules with

antimicrobial and antioxidant activity are excellent immunomodulators (Wanger et al., 1990;

Wadekar,2008).

There are many major roles played by lectins. The development pattern, accumulation

and disappearance of these proteins during germination suggest that lectins may function as

storage proteins (Vanden et al., 1999). Phytolectins have been identified as signal molecule

in Azolla-Anabena symbiosis (Mellor et al., 1981). Lectins have been suggested as a

stimulator of plant embryonic cell (Howard et al., 1972), and have been found to stimulate

pollen germination in Lilium longifolium (Sothworth et al., 1975). Lectins of bulbs of

Eranthus hyenalysis act as an inhibitor of protein synthesis (Kumar et al., 1993). Lectins are

shown to play major role in plant defense system since they have antibacterial (Brockart et

al., 1989), antiviral (Balzarini et al., 1992), and antifungal activity (Brockart et al., 1989).

The phytolectins have been used widely by biomedical scientists to detect sensitization

caused by infectious disease and identification of various blood groups (Boyd and Wax,

1963) and some lectins, even, show the mitogenic and immune stimulatory activities

(Nowell, 1960).

Spleenocytes and lymphocytes are the most important cells of the immune system.

They play an important role in curing blood born and organ born infections. Stimulation of

the spleenocytes and lymphocytes proliferation has been an important aspect of

immunostimulation as it is helpful for curing the infections as early as possible and it is seen

that most of the medicinal plants show immunostimulation by stimulating the proliferation of

spleenocytes and lymphocytes (Saraphanchowitthaya et al., 2007; Butler et al., 2005 and

Exton et al., 2002).


38

From the above mentioned literature survey, the plant lectins have been widely

exploited in almost all the biological fields. The Zizyphus also has tremendous medicinal

properties. However the lectins of this genus are not yet studied for their immunomodulatory

potential. In this context it was decided to isolate, characterize and study the

imunomodulatory activity of the lectins isolated from the of Zizyphus species, the plant

which is widely found in the Nagpur region.

3.2. MATERIALS AND METHODS:

Plant material:

For antibacterial, antifungal, carbohydrate binding and in Immunomodulatory

activity, the purified lectins are used namely ZML-L, ZOL-L, ZJL-L, ZXL-L, ZMS-L, ZOS-

L, ZJS-L and ZXS-L.

Blood Samples:

Human and rabbit blood samples were used as earlier.

Bacterial Culture:

ATCC type cell culture of Gram positive and Gram negative bacteria viz, Escherichia

coli, Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus were obtained from

University Department of Microbiology, RTM Nagpur University, Nagpur.

Fungal Culture:

ATCC type cell culture of fungus viz, Aspergillus flavus, Aspergillus niger,

Aspergillus oryzae, Penicillium chrysogenum were obtained from University Department of

Microbiology, RTM Nagpur University, Nagpur.

Cell lines:

Blood, Lymphnode and Spleen of sheep were obtained from the Animal Husbundry

Department of Veterinary Collage, Nagpur, organs were collected in chilled phosphate buffer

saline and sterilized it by using 70% alcohol before use. The sterilized organs were

trypsinised by trypsin-EDTA solution and cell suspensions were made in culture medium.

Blood was collected in heparinised vial and proliferating blood lymphocytes (PBL’s)

were isolated by using density gradient medium (HiSep LSM, Himedia). PBL’s were isolated

by centrifugation and washed by RPMI -1640, according to the instructions given by the

manufacturer.


39

Culture medium and reagents for cell lines:

Dubecco’s phosphate buffered saline (DPBS), RPMI-1640, Fetal calf serum (FCS)

were purchased from Gibco laboratories. Antibiotic antimycotic solution, phosphate buffer

saline, lipopolysaccharide (LPS), concavlin-A (Con-A), MTT reagent (Tetrazolium dye 3-

[4,5 - dimethylthiazol -2-yl]-2, 5-diphenyl tetrazolium bromide), XTT reagent (Sodium 2,3,-

bis [2-methoxy-4-nitro-5-sulphonyl]-5-[phenylamino] carbonyl -2H- tetrazolium), Zymosan-

A, trypsin, Nitroblue tetrazolium, para- Nitrophenyl phosphate and all the cell culture

medium procured from Sigma Aldrich (St. Louis, USA).

Culture medium for bacterial and fungal culture:

Muller-Hinton agar medium used as a bacterial culture medium. Potato dextrose agar used as

a fungal culture medium. The medium were sterilized and prepared as per the instructions

given by the manufacturer (Himedia, Mumbai).

3.2.1. ANTIBACTERIAL ACTIVITY OF PURIFIED LECTINS OF ZIZYPHUS

SPECIES:

Method:

By using Well Assay method, antibacterial activities of purified protein and their

dilutions fractions of Zizyphus were determined. Four strains of bacteria i.e. Escherichia coli,

Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis were used in the

antibacterial bioassay. Media of MH agar was prepared in conical flask in accordance to the

directions provided by the manufacturer. The media along with Petri dishes, pipette and

metallic borer were sterilized in autoclave for 15 minutes at 121 C and 15 psi pressure. The

media poured into Petri dishes under aseptic condition (Laminar flow hood). Stock solutions

of purified protein 4mg/ ml and their dilutions (1:2, 1:4, 1:8) in PBS were prepared. Positive

control contains antibiotic ampicillin while while for negative control vector solution was

used. After 24 hours antibacterial activities were measured by measuring the diameter of the

zones of inhibition (Ullah et al., 2013).

3.2.2. ANTIFUNGAL ACTIVITY OF PURIFIED LECTIN OF ZIZYPHUS SPECIES:

Method:

By using Well Assay method, antifungal activities of purified protein 4mg/ml and

their dilutions fractions (1:2, 1:4, 1:8,) were determined. Four strains of fungus were used as

mentioned in materials. Media of PDA agar was prepared in conical flask in accordance to


40

the directions provided by the manufacturer. Fungal strains were sub cultured on the agar

media. Stock solutions of purified protein and their dilutions (1:2, 1:4, 1:8) in PBS at

concentration of 4mg/ml were prepared and 1.5, 0.75, 0.325 mg of protein from each stock

solution was added into respective wells. The Petri dishes were incubated in incubator at

37°C for 24 hours and control wells containing antifungal (Clotrimazole) which is a positive

control was also run side by side in the Petri dishes and PBS was used as a negative control.

After 72 hours antifungal activities were measured by measuring the diameter of the zones of

inhibition (Ullah et al., 2013).

3.3.3. CARBOHYDRATE BINDING ACTIVITY OF PURIFIED LECTIN OF

ZIZYPHUS SPECIES

Methods:

Carbohydrate-binding assay were performed by mixing 50 µl of ZML-L, ZOL-L,

ZJL-L, ZXL-L, ZMS-L, ZOS-L, ZJS-L, ZXS-L with an equal volume of 0.1 M different

carbohydrates (D-Arrabinose, α-D-Glucose, D-fructose, D-Mannose, D-Maltose, D-

Galactose, D-Mannitol, Sucrose, Lactose, Raffinose, p-nitrophenyl-α-D-galactopyranoside,

o-nitrophenyl-β-D-galactopyranoside. After 30 min incubation at ambient temperature, 50 µl

of erythrocyte suspension was added and was allowed to settle at room temperature for 1 h

and agglutination was recorded visually. Positive control contained 50 µl of lectins and 50 µl

of normal saline. Negative control contained 50 µl of lectins and 50 µl of erythrocyte

suspension (Kurokawa et al., 1976).

“IN VITRO” AND “IN VIVO” IMMUNOMODULATORY ACTIVITY OF PURIFIED

LECTIN OF ZIZYPHUS SPECIES

3.2.4. METHODS FOR ‘IN VITRO’ IMMUNOMODULATORY ACTIVITY:

Preparation of splenocytes from Sheep:

Spleen of sheep was collected from collected from the Animal Husbundry

Department of Veterinary Collage, Nagpur. Splenocyte suspension was prepared, washed

twice and suspend in complete RPMI 1640 medium (Mansorai et al., 2003). Cell number was

adjusted to1x106 cells/ml.

3.2.4.1. Splenocyte proliferation assay:

Effect of lectin on splenocyte proliferation was tested by XTT assay (Kainthala et al.,

2006). Spleenocyte suspension (1x106 cells/well) suspended in complete RPMI 1640

medium were treated with different concentration of lectins (starting from the effective dose


41

decided by the MTT assay and dilutions) dissolved in PBS. The PBS was used as a control

while atropine solution was taken as a negative control. One group was treated with 10 µg/ml

LPS (a powerful mitogen). After incubation for 48 h at 37 oC in 5% CO2 humidified

atmosphere, the medium was removed and the adherent macrophages were washed twice

with RPMI medium. A mixture of XTT and PMS (N-methyl dibenzopyrazine methyl

sulphate) was then introduced along with RPMI 1640 medium. Cells were then incubated for

4 h at 37 oC in 5% CO2 humidified atmosphere. Absorbance was measured at 450 nm using

microplate reader. Splenocyte proliferation stimulation index (SSI) was calculated as:

SSI = O. D. of Experimental / O. D. of control

Preparation of lymphocytes from Sheep:

Sheep lymphnodes were collected from the Animal Husbundry Department of

Veternary Collage, Nagpur. and lymphocytes suspension was prepared, washed twice and

suspend in complete RPMI 1640 medium (Mansorai et al., 2003). Cell number was adjusted

to 1x106 cells/ml.

3.2.4.2. Lymphocyte proliferation assay:

Effect of lectin on splenocyte proliferation was tested by XTT assay almost similarly

as that of spleenocyte proliferation. Lymphocyte proliferation stimulation index (LSI) was

calculated as:

LSI = O. D. of Experimental / O. D. of control

3.2.4.3. Phagocytic Index Assay:

Capacity of lectins to stimulate phagocytosis was tested by NBT dye reduction assay

(Rainard, 1986). Macrophages (1x106 cells/well) suspended in complete RPMI 1640 medium

were treated with different concentration of lectins (starting from the effective dose decided

by the MTT assay and dilutions) dissolved in PBS. Control was run with PBS (without

lectins). After incubation for 24h at 37 oC in 5% CO2 humidified atmosphere, medium was

removed and adherent macrophages were washed twice with RPMI medium. Zymosan – A

(1µg/ml of PBS) was introduced along with NBT solution (1.5 mg/ml of PBS) and cells were

incubated for 60 min at 37 oC in 5% CO2 humidified atmosphere (Thermo Scientific, Class-

100). After 60 min, medium was removed and cells were washed twice with PBS and air

dried. Finally, 2 M KOH was added and absorbance was measured at 570 nm using micro

plate reader (Thermo electron corp., 358) Phagocytic index (PI) was calculated by following

equation:

PI = O. D. of Experimental / O. D. of control

3.2.4.4. Lysosomal enzyme activity assay:


42

Ability of lectins to stimulate lysosomal enzyme activity (acid phosphatase) was

tested using p-NPP (p-nitrophenyl phosphate) assay (Suzuki et al., 1988). Macrophages

(1x106cells/well) suspended in complete RPMI 1640 medium were treated with different

concentration of lectins in µg/ml dissolved in PBS. Control was run with PBS (without

lectins). After incubation for 24h at 37 oC in 5% CO2 humidified atmosphere, medium was

removed and adherent macrophages were washed twice with RPMI medium. 1 % Triton X-

100, 10 mM p-NPP and 0.1 M citrate buffer (pH 5.0) were then introduced and cells were

incubated for 37 oC in 5% CO2 humidified atmosphere. After 30 min, 0.2 M borate buffer

(pH 9.8) was added and absorbance was measured at 405 nm using microplate reader.

Lysosomal enzyme activity index (LI) was calculated according to equation:

LI = O. D. of Experimental / O. D. of control

3.2.5. METHODS FOR ‘IN VIVO’ IMMUNOMODULATORY ACTIVITY:

Animals:

Wistar albino rats (200 – 250 g) of both sex were procured from National Center for

laboratory Animal Sciences, Hyderabad, India. Animals were maintained under the standard

conditions (temperature 25±2oC) with 12 h light/ 12 h dark cycle. They were fed ad libitum

with standard pallet diet and purified water. Human care was provided to all animals and

norms prescribed by Animal ethics committee were critically followed (Permission letter

from animal ethics committee dated: 19/ 08/ 2013, Annexure - I). During oral administration,

care was taken to prevent any injury to animals. Animals were weighed on electronic

weighing balance (Essae Teraoka Ltd. FB2200) before experiments.

3.2.5.1. Acute toxicity study:

A group of six wistar rats was daily orally administered with lectins. In the first week,

2 mg/kg of body weight (bw) dose was provided and animal behavior was regularly

observed. As no change in behavior was observed, dose was shifted to 5 mg/ kg of bw. Thus

graded doses of 50, 75, 100, 200, 250, 500, 1000, 2000 and 4000 mg/kg of bw were

successively provided for a week. Half of lethal dose LD50 as obtained from this experiment

was 2000 mg/kg bw at which rats showed little bit different behavior than regular. Dose

selection was performed by taking 1/10th of LD50 value. Thus, for in vivo studies, dose of

lectin selected was 200mg/ kg bw.


43

3.2.5.2. Cytokine assay (TNF-α) and Nitric oxide (NO) Estimation:

A group of six Wistar albino rats was daily orally administered with lectins for 28

days as per the value obtain by the toxicity assay, in experimental group of four different

seed lectins and control group orally administered with normal saline. On the next day blood

withdrawn by the orbital plexus of rat and serum was separated as a source of cytokines and

for nitric oxide estimation. The cytokine assay was performed by the kit method as per the

instruction given by the manufacturer. (Invitrogen Corporation, Camarillo, CMC3013,

837111). The concentration of NO was determined by Colorimetric Nitric Oxide Assay

(Miranda et al., 2001).

3.2.5.3. Hemagglutination Assay:

Six rats were taken in each group (2 month old; average weight 200-225 g). Animals

were devided into three groups; positive Control (PC), Negative control (NC) and Four

Experimental (E) (for each: ZMS, ZOS, ZJS, ZXS). Sheep RBC’s (SRBC) were collected

from the Animal Husbandry Department of Veterinary Collage, Nagpur. Sheep blood was

withdrawn from external jugular vein of sheep with intravenous set and was directly

introduced in Alsvier solution in 1:1 proportion with gentle mixing (John, 2010). Lectin were

administered orally in in PBS (10 mg/kg bw) to NC and all E from day -10 (10 days prior to

sensitization injection). Intravenous injection (sensitization) of 0.5 x 106 SRBCs in 0.2 ml

PBS was administered to PC and all E on day 0. Lectin treatment was continued till day +15

to NC and all four E. Blood was withdrawn from orbital plexus of all groups of rats on day

+15. Serum was considered as a source of antibodies against SRBCs. Different dilutions of

all groups of sera were prepared and checked for hemagglutination assay by titrating serum

dilutions with SRBCs (0.025 x 106 cells / ml). Microtitre plates were incubated at room

temperature for 2 h and examined visually for agglutination. Reciprocal of the highest

dilution of serum showing 50% agglutination was expressed as HA titre (Mitra et al., 1999).

3.2.5.4. Effect of lectins in preventing systemic anaphylactic shock:

Total 6 rats were included in each group (2 months old with average weight 200-225

g). Animals were divided into four groups; positive control (PC), negative control (NC),

experimental 1 (E1) (for each: ZMS, ZOS, ZJS, ZXS) and experimental 2 (E2) (for each:

ZMS, ZOS, ZJS, ZXS). Oral administration of lectin was given by dissolving in PBS. Lectins

were administered (200 mg/kg of bw) to E1 daily from day-10 (10 days prior to sensitization

injection). Intraperitonial injection (sensitization) of BSA (1 mg in 0.2 ml PBS) was

administered to all four groups on day 0. Lectin treatment was continued till day +15 to E1.

Intravenous injection (shocking injection) of BSA (1 mg in 0.2 ml PBS) was given to PC and

E1 while NC was treated with intravenous injection of ovalbumin (1 mg ovalbumin in 0.2 ml

PBS). To E2, along with intravenous shocking injection of BSA, 1 mg lectin was injected in


44

combination. Systemic anaphylactic reaction was observed within 10 min after shocking

injection and rated as: Positive reaction; animals died or rendered stationary for at least 1

min, Negative reaction; no changes were observed in activity and movement of animals was

normal (Hsu et al.,1997).

3.2.5.5. Arthus Reaction:

Total 6 rats were included in each group (2 months old with average weight 200-225

g). Animals were divided into four groups; positive control (PC), negative control (NC),

experimental 1 (E1) (for each: ZMS, ZOS, ZJS, ZXS) and experimental 2 (E2) (for each:

ZMS, ZOS, ZJS, ZXS). Oral administration of lectin was given by dissolving in PBS. Lectins

were administered (200 mg/kg of bw) to E1 daily from day-10. Subcutanious injection of 1

mg BSA in 0.2 ml PBS was given to all groups on day 0 (sensitization). Lectin treatment was

continued till day +15 to E1. Intradermal injection (shocking injection) of 0.5 mg BSA in 0.2

ml PBS was given to PC and E1 in right foot pad while to NC as a shocking injection; an

intradermal injection of ovalbumin (0.5 mg ovalbumin in 0.2 ml PBS) was injected in right

foot pad. Immediate effectiveness of lectin on arthus reaction was tested in E2, where along

with shocking injection of BSA, 1 mg lectin was injected in combination. Thickness of

footpad of each rat was recorded from all different angles by venire caliper at 2 h, 4 h, 6 h,

1day, 2 days, 3 days after shocking injection (Hsu et al.,1997).


45

3.3. RESULTS:

3.3.1. Antibacterial activity of purified lectins of Zizyphus species:

No zone of inhibition was found after 24 h incubation at 37 oC, but the bacteriostatic

activity was seen upto 18 h. The clumps of bacteria were seen on the microscopic slide when

lectin solutions were mixed with bacteria.

Table 3.1 Antibacterial activity of purified lectins from leaves and seeds of Zizyphus

species

Sr. No.

Lectin samples

Name of Bacteria Observations Results

1 ZML-L Escherichia coli No zone of inhibition was found Bacteriostatic

Pseudomonas aeruginosa No zone of inhibition was found Bacteriostatic

Staphylococcus aureus No zone of inhibition was found Bacteriostatic

Bacillus subtilis No zone of inhibition was found Bacteriostatic

2 ZOL-L Escherichia coli No zone of inhibition was found Bacteriostatic




3 ZJL-L Escherichia coli No zone of inhibition was found Bacteriostatic




4 ZXL-L Escherichia coli No zone of inhibition was found Bacteriostatic




5 ZMS-L Escherichia coli No zone of inhibition was found Bacteriostatic




6 ZOS-L Escherichia coli No zone of inhibition was found Bacteriostatic




7 ZJS-L Escherichia coli No zone of inhibition was found Bacteriostatic




8 ZXS-L Escherichia coli No zone of inhibition was found Bacteriostatic





46

3.3.2. Antifungal activity of purified lectins of Zizyphus species:

No zone of inhibition was found after 72 h incubation at 30 oC, but the fungistatic

activity was seen upto 48 h. The clumps of fungal spores were seen on the microscopic slide

when lectins solution mixed with spores of the fungus.

Figure 3.2 Antifungal activity of purified lectins from leaves and seeds of Zizyphus

species

Sr. No.

Lectin samples

Name of Fungus Observations Results

1 ZML-L Aspergillus flavus No zone of inhibition was found Fungistatic

Aspergillus niger No zone of inhibition was found Fungistatic

Aspergillus oryzae No zone of inhibition was found Fungistatic

Penicillium chrysogenum No zone of inhibition was found Fungistatic

2 ZOL-L Aspergillus flavus No zone of inhibition was found Fungistatic




3 ZJL-L Aspergillus flavus No zone of inhibition was found Fungistatic




4 ZXL-L Aspergillus flavus No zone of inhibition was found Fungistatic




5 ZMS-L Aspergillus flavus No zone of inhibition was found Fungistatic




6 ZOS-L Aspergillus flavus No zone of inhibition was found Fungistatic




7 ZJS-L Aspergillus flavus No zone of inhibition was found Fungistatic




8 ZXS-L Aspergillus flavus No zone of inhibition was found Fungistatic





47

Table 3.2 Antifungal activity of standard antifungal agent

Sr. No. Fungal Culture Standard Anibiotics Zone of inhibition after 72 h

1 Aspergillus flavus Chlotrimazole 18 mm

2 Aspergillus niger Chlotrimazole 12 mm

3 Aspergillus oryzae Chlotrimazole 14 mm

4 Penicillium chrysogenum Chlotrimazole 22 mm

3.3.3. Carbohydrate binding activity of purified lectins of Zizyphus species:

The carbohydrate-binding specificity of above lectin was estimated by the ability of

series of single sugars to inhibit the hemagglutination of the rabbit erythrocytes. To

characterize the carbohydrate binding specificity of purified lectins, inhibition experiments

were performed. Examination of the results were plotted in the Figure, 3.3 – 3.10. The level

of inhibition by various carbohydrates of the hemagglutinating activity against rabbit

erythrocytes was assessed in order to determine the carbohydrate affinity of the lectins. It

was observed that a carbohydrate containing D- galactosel was able to bring about total

inhibition of hemagglutination. These results indicate that the purified lectins are galactose-

binding lectins.


50

3.3.4 ‘IN VITRO’ AND ‘IN VIVO’ IMMUNOMODULATORY ACTIVITY

IN VITRO IMMUNOMODULATORY ACTIVITY

3.3.4.1. Cytotoxic activity by MTT assay:

The effective dose for splenocytes, lymphocytes and peripheral blood lymphocytes

(PBL’s) was determined by the calorimetric MTT assay kit (Himedia, Mumbai, india). The

cell suspention was 1 x106 / ml and different concentration of lectin was added. After 48 h

incubated in humidified 5% CO2 incubator the MTT assay was performed as per the

instruction given by the manufacturer of MTT kit and effective dose was determined which is

as given below;

Table 3.3 Effective dose of purified lectins estimated by MTT assay for different cell types.

Sr.

No.

Name of Protein Sample Type of Cells

(1 x106 / ml)

Effective Dose of

Lectins (µg/0.1ml)

1 Zizyphus mauritiana leaf lectin PBL’s 12.5

Lymphocytes 12.5

Splenocytes 25

2 Zizyphus oenoplia leaf lectin PBL’s 25

Lymphocytes 50

Splenocytes 12.5

3 Zizyphus jujuba leaf lectin PBL’s 12.5

Lymphocytes 12.5

Splenocytes 25

4 Zizyphus xylopyra leaf lectin PBL’s 12.5 Lymphocytes 12.5

Splenocytes 25

5 Zizyphus mauritiana seed lectin PBL’s 12.5 Lymphocytes 50

Splenocytes 50

6 Zizyphus oenoplia seed lectin PBL’s 12.5 Lymphocytes 50

Splenocytes 50

7 Zizyphus jujuba seed lectin PBL’s 25

Lymphocytes 100 Splenocytes 50

8 Zizyphus xylopyra seed lectin PBL’s 25

Lymphocytes 100 Splenocytes 50


51

3.3.4.2. Splenocyte proliferation assay:

Lectins were found to be quite effective in stimulating splenocyte proliferation. The

stimulation index at effective dose of each lectin found by MTT assay previously, shows

little less than the LPS and near to the activity of Con – A which are very effective known

mitogens (Figure, 3.11 – 3.18). Atropine in the negative control was unable to induce any

proliferation in splenocytes.

3.3.4.3. Lymphocyte proliferation assay:

As for splenocytes, lectins were found to be effective in lymphocyte proliferation too.

At an effective dose determined by the MTT assay, the lectins gave a maximum proliferation

little less than the LPS and comparable with Con - A which are known mitogen. The

Atropine present in the negative control as usual was found to be ineffective (Figure, 3.19 –

3.26).

3.3.4.4. Phagocytic Index Assay:

Purified lectins from leaves and seeds of the zizyphus species was explored for its

phagocytosis stimulating activity at different concentrations. The first concentration was

calculated by the MTT assay, where maximum proliferation of the blood lymphocytes was

observed for all the leaves and seed’s lectin samples. Lectins were explored for its

phagocytosis activity at different concentrations by dissolving it in PBS. It was observed that

the effective concentration has shown its maximum activity (Figure, 3.27 – 3.34). Atropine, a

standard alkaloid used as negative control did not show any considerable activity.

3.3.4.5. Lysosomal enzyme activity assay

The lectins show dose dependent increase in the activity on lysosomal enzymes. At

maximum effective concentration determined by the MTT assay, the lysosomal the

lysosomal enzyme activity has shown an increase of more than double as compared to

controls (Figure, 3.35 – 3.42). The Atropine used as a negative control shows no effect on

enzyme activity.


In the figures C was compared with all different dilutions of lectins and NC where, *: p ≤ 0.0001, **: p ≤ 0.005, ***: p ≤ 0.05 and #: p ≥ 0.05.


In the figures C was compared with all different dilutions of lectins and NC where, *: p ≤ 0.0001, **: p ≤ 0.005, ***: p ≤ 0.05 and #: p ≥0.05.


61

“IN VIVO” IMMUNOMODULATORY ACTIVITY OF PURIFIED LECTIN OF

ZIZYPHUS SPECIES

3.3.4.6. Cytokine assay (TNF-α) and Nitric oxide (NO) Estimation:

The cytokine TNF- α and nitric oxide level was found to be normal in the control rats while

decreased or still up to normal in the groups which had oral treatment of seed lectins. It was

found that the oral treatment of lectins in the Wistar albino rats was helpful to normalize the

level of TNF- α and nitric oxide comparable to the control group (Figure, 3.43 & 3.44).

In the figures C was compared with all different dilutions of lectins and NC where, *: p ≤ 0.0001, **:

p ≤ 0.005, ***: p ≤ 0.05 and #: p ≥0.05. Data is express as mean (SD) where n = 6.


62

3.3.4.7. Haemagglutination assay:

Result presented in figure 3.45 depict the effect of purified seed lectins, ZMS-L,

ZOS-L, ZJS-L and ZXS-L on humoral immunity through antibody formation in wistar albino

rats. Animals in positive control group (PC) sensitized with SRBCs intravenously at day 0

had shown HA titre. In negative control group (NC), only 25 oral supplementation of lectin

were given while intravenous sensitization was given. Hence this group did not show any

antibody formation. In all experimental group (E), after 25 oral supplement of lectins,

including 10 doses before SRBC sensitization, animals shows increased antibody titer by the

effect of lectin in stimulating antibody formation.

As compared to the positive control (PC), experimental (E) of ZMS-L and ZXS-L

shows great increase in antibody formation than ZOS-L and ZJS-L. In case of NC (Negative

control) as no sensitizing injection was given, no antibody (no titer) formation was seen.

Data is shown as mean value where n=6.

3.3.4.8. Effect of lectins in preventing systemic anaphylactic shock:

Results presented in table 3.4 – 3.7 depict the effect of lectins in preventing

anaphylactic shock in wistar rats. All animals in PC displayed symptoms of systemic

anaphylactic shock (animal remained stationary at least for 1 min) while NC did not show

any anaphylactic reaction. In E1 after 25 oral supplementations of lectins (ZMS-L, ZOS-L,

ZJS-L and ZXS-L), animals did not show systemic anaphylaxis. An immediate effect of

lectins on anaphylactic shock was however, not apparent when injected in combination with

BSA in shocking injection to E2.


63

In case of PC, all 10 rats had shown anaphylactic shock as compared to E1 where

neither of the rats was recorded with anaphylactic shock. However, in case of negative

control as shocking injection was replaced with ovalbumin instead of BSA, no anaphylactic

shock was observed. While in case of E2, immediate effect of lectin was seen.

Table 3.4 Effect of ZMS-L on the inhibition of active systemic anaphylaxis in rats

Sensitizing

injection

Shocking

injection

Oral ZMS-L

treatment

Results

S/T D/T

PC BSA (i.p.) BSA (i.v.) - 10/10 0/10

NC BSA (i.p.) OVA (i.v.) - 0/10 0/10

E1 BSA (i.p.) BSA (i.v.) + 0/10 0/10

E2 BSA (i.p.) BSA (i.v.) + ZMS-L (i.v.)

- 0/10 0/10

Table 3.5 Effect of ZOS-L on the inhibition of active systemic anaphylaxis in rats

Sensitizing

injection

Shocking

injection

Oral ZMS-L

treatment

Results

S/T D/T

PC BSA (i.p.) BSA (i.v.) - 10/10 0/10

NC BSA (i.p.) OVA (i.v.) - 0/10 0/10

E1 BSA (i.p.) BSA (i.v.) + 0/10 0/10

E2 BSA (i.p.) BSA (i.v.)

+ ZOS-L (i.v.)

- 0/10 0/10

Table 3.6 Effect of ZJS-L on the inhibition of active systemic anaphylaxis in rats

Sensitizing

injection

Shocking

injection

Oral ZMS-L

treatment

Results

S/T D/T

PC BSA (i.p.) BSA (i.v.) - 10/10 0/10

NC BSA (i.p.) OVA (i.v.) - 0/10 0/10

E1 BSA (i.p.) BSA (i.v.) + 0/10 0/10

E2 BSA (i.p.) BSA (i.v.)

+ ZJS-L (i.v.)

- 0/10 0/10

Table 3.7 Effect of ZXS-L on the inhibition of active systemic anaphylaxis in rats

Sensitizing

injection

Shocking

injection

Oral ZMS-L

treatment

Results

S/T D/T

PC BSA (i.p.) BSA (i.v.) - 10/10 0/10

NC BSA (i.p.) OVA (i.v.) - 0/10 0/10

E1 BSA (i.p.) BSA (i.v.) + 0/10 0/10

E2 BSA (i.p.) BSA (i.v.) + ZXS-L (i.v.)

- 0/10 0/10

For Table 3.4 – 3.7: , n=6

PC=positive control, NC= negative Control, E1=Experimental 1,E2=Experimental 2

OVA= Ovalbumin, i.p.= intraperitonial, i.v.=intravenous;

S/T= Number of anaphylactic symptoms/total no. of rats;

D/T= Number of anaphylactic deaths/total no.of rats


64

3.3.4.9. Effect of lectins in preventing Arthus reaction:

All animals in PC displayed positive footpad reaction within 2 h of shocking injection

(Figure, 3.46). However, in NC, rats did not show arthus reaction. After 25 oral

supplementation of lectins to E1, no Arthus reaction observed in the right footpad. In PC, the

right footpad shown an increase of approximately 12 mm was recorded within 2 h as

compared to E1 where an increase if just 6 mm was recorded. Thus, severity of arthus

reaction was found to decrease to nearly upto normal (Figure, 3.47 – 3.50). However in E2,

lectins was found to be comparatively less effective in inhibiting arthus reaction instantly but

has decreased the severity of footpad reaction after taking more time as compared to PC.

Figure 3.46 Photograph showing foot pad reaction in Wistar rats

Figure: A Figure :B

Figure:A; Positive Arthus reaction in PC, Figure:B; Prevention of Arthus reaction due to lectin in E1

In case of PC, positive Arthus reaction was observed within 2 h while in case of E1, prevention of the

footpad reaction was observed. In E1, no significant difference was observed between left and right

footpad after 2 h.

PC= Positive Control, E1= Experimental 1


In the figures C was compared with E1 and E2 where, *: p ≤ 0.0001, **: p ≤ 0.005, ***: p ≤ 0.05 and #: p ≥ 0.05, n=6


In the figures C was compared with E1 and E2 where, *: p ≤ 0.0001, **: p ≤ 0.005, ***: p ≤ 0.05 and #: p ≥ 0.05, n=6.


67

3.4 CONCLUSION:

From the result it was found that the purified lectins do not have antibacterial and

antifungal activity, but it have bacteriostatic and fungistatic activity.

Lectins have shown to be great impact in stimulating phagocytosis in a dose

dependent manner. These results were supported by lysosomal enzyme activity assay which

indicate immunostimulation in positive direction. Mitogenic potential of lectin was clear

from its ability to stimulate splenocytes and lymphocytes. It was found to be effective near to

known mitogen as LPS and Con-A. In all in vitro parametes, atropine was taken as negative

control as it is alkaloid in nature, it dosent possess immunostimulatory activity (Yieng-zhe et

al.,2006).

Nitric oxide (NO) is one of the few gaseous signaling molecules known (Umar and

Laarse, 2010). It is involved in many physiological and pathological processes within the

body, both beneficial and detrimental (Kruger and Linke, 2009; Sesa, 2009). Appropriate

levels of NO production are important in protecting organs from ischemic damage (Krauss et

al., 2009).

Cytokines are believed to be important in the pathogenesis of decompensate alcoholic

liver disease, end-organ failure and injuries (Tilg and Diehl, 2000). A predominantly

proinflammatory cytokine profile might cause hepatic inflammation and necrosis.

Mitochondria are a target for tumor necrosis factor (TNF) initiated death signals, releasing

reactive oxygen species, leading to apoptosis and cell death (Higuchi et al., 1997 and

Schulze-Osthoff et al., 1992). Inflammation and oxidative stress also induce production of

nitric oxide (NO), which appears to cause the circulatory (Vallance et al., 1991) and renal

(Gines et al., 1993) disturbances of liver failure. There is increasing evidence that the

mediators of inflammation (e.g., proinflammatory cytokines, NO, and oxidative stress) could

modulate (Jalan and Williams, 1993),the effect of hyperammonaemia (Kramer et al., 2000)

in precipitating encephalopathy. Both TNF- α and NO is the indicators of inflammation and

poses increased oxidative threat to the exposed subjects.

In the present investigation rats showed a marked normal level without any increase

in the levels of TNF-α and NO in the serum clearly signify the lectin itself didn’t induce an

inflammation or oxidative stress by which it can cause any injury. Administration of lectin in

rats exhibited a significant normal or somehow reduction in the levels of serum TNF-α and

NO which shows the anti-inflammatory ability of isolated lectins from Zizyphus species.


68

Many immune stimulators are effective in vitro but when tested in vivo they do not

manifest the desirable results. But the lectins has proved its potential by elevating antibody

formation in Wistar rats. After regular doses of lectins, HA titer was found to get doubled

and more than doubled in some cases. Thus lectins have successfully stimulated acquired

immunity through antibody formation. Lectins have raised the concentration of immune cells

in Wistar rats. In the results presented in anti- allergic activity the lectins however, very

beautifully prevent the anaphylactic shock without any mortality and Arthus reaction. Many

immunodeficiency diseases like AIDS, these lectins can be a perfect replacement for

synthetic immunostimulators or it can be given as a supplement along with drugs. Thus lecin

have great potential to stimulate both innate and acquired immunity.

Anaphylaxis is a serious, life-threatening situation which involves multiple organs

and usually found rapid in onset. At its most severe condition, there are symptoms like

broncho-spasm, airway angioedema and hypotensive shock. It is an IgE mediated type 1

hypersensitivity reaction involves activation of mast and release of as histamine,

leukotrienes, TNF and various other cytokines. Anaphylaxis is the most serious and life-

threatening form of systemic allergic reactions. The results show that after oral treatment of

isolated lectins in Wistar albino rats did not indicate any symptoms of anaphylaxis and

mortality.

The Arthus reaction happens due to the in situ formation of antigen/antibody

complexes after the intradermal entry of an antigen. If the animal/patient was previously

sensitized, an Arthus reaction occurs. Typical of most mechanisms of the type III

hypersensitivity, manifestation of Arthus as local vasculitis due to deposition of IgG-based

immune complexes in dermal blood vessels. The end result is a localized area of redness and

induration that typically lasts a day or so. In the present investigation of Arthus reaction the

sevearity of inflammation was observed in the inflammation of footpad of wistar albino rat.

The oral treatment of lectins did decrease the swelling of footpad as compared to

subcutaneous treatment along with time.

As per the result presented in this report lectins were isolated and purified by the

standard purification procedures including ammonium sulphate precipitation, affinity

chromatography on cross linked guar-gum and ion exchange chromatography on DEAE-

cellulose. The isolated lectins were found to be monomeric and low molecular weight on

native and SDS polyacrylamide gel electrophoresis. Purified lectins have sugar specificity

towards D-galactose and its derivatives, lectins were inhibited by the heavy metal ions like

Hg++, Pb++ and Sb++ and activity was restored by Mn++, Mg++ and Ca++. Purified lectins

were found to be optimally active at pH 7and active from the pH range 4 - 10 and

thermostable. From the results presented in this report, it can be concluded that lectins

isolated from Zizyphus species were found to be very efficient stimulator of both innate and

acquired immune system, enhance phagocytic index, lymphocyte proliferation, splenocyte


69

proliferation lysosomal degranulation shows stimulated cellular immunity while increased

hemagglutination titre shows elevated humoral immunity. Lectins were found to be efficient.

Being natural, chances of side effect are less. To the best of our knowledge present report is

the first of our kind on isolation, characterization and Immunostimulatory activity of lectins

from these four Zizyphus species of Maharashtra.


70

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