POLYSACCHARIDES FROM Sebdenia polydactyla AND Oryza …shodhganga.inflibnet.ac.in/bitstream/10603/21972/2/2 thesis.pdf · Polysaccharides are carbohydrates that form the bulk of all

POLYSACCHARIDES FROM

Sebdenia polydactyla AND Oryza sativa: STRUCTURAL FEATURES, CHEMICAL MODIFICATION AND

ANTIVIRAL ACTIVITIES

THESIS SUBMITTED FOR THE DEGREE OFDOCTOR OF PHILOSOPHY (SCIENCE) IN CHEMISTRY

OF THE UNIVERSITY OF BURDWAN2012

TUHIN GHOSH, M.Sc.

NATURAL PRODUCTS LABORATORYDEPARTMENT OF CHEMISTRY

THE UNIVERSITY OF BURDWANWEST BENGAL, INDIA

THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (SCIENCE) IN CHEMISTRY

OF THE UNIVERSITY OF BURDWAN 2012

POLYSACCHARIDES FROM

Sebdenia polydactyla AND Oryza sativa: STRUCTURAL FEATURES,

CHEMICAL MODIFICATION AND ANTIVIRAL ACTIVITIES

TUHIN GHOSH, M.Sc.

NATURAL PRODUCTS LABORATORY DEPARTMENT OF CHEMISTRY

THE UNIVERSITY OF BURDWAN WEST BENGAL, INDIA

Dedicated

To

My Mentor,

Prof. Bimalendu Ray

And

Late Mrs. Shefali Ghosh

Learning without thought is labor lost; thought without learning is perilous. ----------- Confucius, Analects

THE UNIVERSITY OF BURDWAN

Dr. Bimalendu Ray Associate Professor

****

Department of Chemistry Golapbag, Burdwan 713104, India Tel: +91-342-2533913 (O) +91-342-2657709 (R) Fax: +91-342-2530452 (O) E-mail: [email protected] Dated…………………..

TO WHOM IT MAY CONCERN

This is to certify that the thesis “POLYSACCHARIDES FROM

Sebdenia polydactyla AND Oryza sativa: STRUCTURAL FEATURES, CHEMICAL

MODIFICATION AND ANTIVIRAL ACTIVITIES” is the result of work done by

Mr. Tuhin Ghosh, M.Sc., who has registered his name in The University of

Burdwan on 12-10-2007 for the award of Doctor of Philosophy (Science) in

Chemistry under my supervision and guidance. It is also certified that this

work has not been submitted for any degree by Mr. Tuhin Ghosh or other. It is

worth mentioning that Mr. Ghosh has successfully completed Ph.D.–course

work framed by the Department of Chemistry, following the guidelines of The

University of Burdwan, and that he has delivered one seminar lecture on 08-

02-2012 in our department regarding fulfillment of all the requirements for

submitting the thesis for Ph.D. degree under new regulation, 2009 of this

University.

Dr. Bimalendu Ray

Supervisor

ACKNOWLEDGEMENT

I would like to take this opportunity to express my deep sense of gratitude and respectful

regards to my supervisor and mentor Dr. Bimalendu Ray, Associate Professor, Department of

Chemistry, The University of Burdwan for his untiring help and encouragement and stimulating

guidance through the course of this work.

I shall be failing in my duties if I do not express my heartfelt respects to Prof. Pradyut K

Ghosal, Prof. Subrata Laskar, Prof. Barindra K Ghosh and Dr. Pabitra Chattopadhyay, the

present Head of the Department of Chemistry, The University of Burdwan for their enthusiastic

co-operation and constant encouragement.

I record my indebtedness to Prof. Elsa B Damonte, Cindad Universitaria, Argentina and

to Prof. Manfred Marschall, University of Erlangen-Nuremberg, Erlangen, Germany, for their

help with Bio-assay.

I am grateful to all the faculty members, staffs, and research scholars of the Department

of Chemistry, The University of Burdwan, for their kind cooperation in academic, official and

laboratory works related to this thesis. Valuable help rendered by lab-mates Dr. Utpal Adhikari,

Dr. Kausik Chattopadhyay, Dr. Pinaki Mandal, Mrs. Paramita Karmakar, Ms. Nabanita

Chattopadhyay, Ms. Sharmistha Sinha, Mr. Sudipta Saha, Mr. Shruti Saurabh Bandyopadhyay,

Ms. Debjani Ghosh, Mr. Udipta R Chatterjee and Mr. Sujay Majee deserves special mention.

I am also appreciative of and thankful to my parents, Mr. Gobinda Ch Ghosh and Mrs.

Kalyani Ghosh and my wife, Mrs. Ankita Basu whom I adore most for their love and sacrifices,

forming the very platform of my life. My sincere thanks are cordially extended to Mrs. Tapashi

Ray, Mrs. Maya Ray and Ms. Sayani Ray for their enthusiastic encouragement and cooperation.

Financial assistance received from Council of Scientific and Industrial Research and

Department of Science and Technology, New Delhi through Sanction No. 09/025(0162)/2007-

EMR-I and SR/S1/OC-50/2007, respectively and the infrastructural facility from The University

of Burdwan are gratefully acknowledged.

Department of Chemistry

The University of Burdwan Tuhin Ghosh Burdwan, 713104, India

PREFACE

Polysaccharides are carbohydrates that form the bulk of all food consumed by humans

and that are essential for the maintenance of life and good health. During the last two decades,

much attention has been focused on the pharmacological activities of many naturally occurring

polysaccharides. The possible role of these biopolymers for treating a broad varieties of diseases

such as tumor, thrombosis, virus-related diseases, diabetes, ulcer etc. constitute the subject

matter of several excellent reviews, yet the relationship between the chemical structure of

polysaccharides and their physical activities is not very clear.

The present work is an endeavor to investigate structural features of sulfated

polysaccharides from Sebdenia polydactyla and Oryza sativa. Chapter 1 reports about

Herpesviridae family, the structure of the viral glycoproteins and cell surface receptors, the

clinically approved anti viral drugs, and sulfated polysaccharides as anti viral drug candidates.

Chapter 2 describes water soluble sulfated polysaccharides present in Sebdenia polydactyla.

Purification of polysaccharides and determination of their structure and anti herpetic activities

constitute the major part of this chapter. Chapter 3 provides information on the molecular

structures of glucans and the anti cytomegalovirus activity of a series of sulfated glucans

generated from a commercial preparation of rice bran (Oryza sativa). Chapter 4 describes

general methods used in the structure determination and in the determination of anti viral

activities.

Department of Chemistry

The University of Burdwan Tuhin Ghosh

Burdwan – 713104, India

INTRODUCTION

1-1. HERPES SIMPLEX VIRUS INFECTIONS 2

1-2. THE RECEPTOR-BINDING SITE OF VIRAL 3

GLYCOPROTEIN

1-3. CELL-SURFACE GLYCOSAMINOGLYCAN 5

(GAG) RECEPTORS

1-4. ANTI-VIRAL DRUGS: THE TREATMENT OF 6

HERPES VIRAL INFECTIONS

1-5. SULFATED POLYSACCHARIDES AS ANTI-VIRAL 9

AGENTS

STRUCTURAL FEATURES AND ANTI VIRAL ACTIVITY OF SULFATED XYLOMANNANS FROM THE RED SEAWEED Sebdenia polydactyla

2-1. INTRODUCTION 15

2-2. CHEMICAL CHARACTERIZATION OF SULFATED 16

XYLOMANNANS FROM Sebdenia polydactyla

2-2.1. Preparation of depigmented algal power and sugar 16

composition analysis 2-2.2. Isolation and sugar composition of polysaccharide fractions 18

2-2.3. Purification of the sulfated xylomannans by chromatography 20

2-2.4. Desulfation and further sulfation 21

Contents

AIM OF THE THESIS 13

CHAPTER 2

CHAPTER 1

2-2.5. Infra red (IR) spectroscopy 22

2-2.6. Molecular mass 23

2-2.7. Smith degradation 24

2-2.8. Glycosidic linkage analysis 24

2-2.9. Nuclear magnetic resonance (NMR) spectroscopy 28

2-3. BIOLOGICAL ACTIVITIES OF SULFATED 30


2-3.1. Cytotoxicity 30

2-3.2. Anti herpetic activities 31

2-4. CONCLUSIONS 34

ANTI CYTOMEGALOVIRUS ACTIVITY AND STRUCTURAL FEATURES OF SULFATED GLUCANS PREPARED FROM A COMMERCIAL SOURCE OF RICE BRAN (Oryza sativa)

3-1. INTRODUCTION 37

3-2. CHEMICAL CHARACTERIZATION OF GLUCANS 39

3-2.1. Preparation of de oiled rice bran and sugar composition analysis 39 3-2.2. Isolation and degradation 41

3-2.3. Generation and purification of sulfated glucans 43

3-2.4. Molecular mass 44

3-2.5. IR-Spectroscopy 45

3-2.6. Linkage analysis 46

3-2.7. NMR spectroscopy 51

3-3. BIO-ASSAY OF RICE BRAN DERIVED SULFATED 53

GLUCANS

3-3.1. Anti cytomegalovirus activity 53

CHAPTER 3

3-3.2. Viral entry is the main mode of activity of sulfated glucans S1G 55

and S2G

3-3.3. Antiviral activity of S1G is selectively detectable for human 58

cytomegalovirus but not for other human herpesvirus

3-4. CONCLUSIONS 59

MATERIALS AND METHODS

4-1. PLANT MATERIALS AND PRELIMINARY TREATMENTS

4-1.1. Red seaweed Sebdenia polydactyla 62

4-1.2. Commercial preparation of rice bran (Oryza sativa) 62

4-2. EXTRACTION OF POLYSACCHARIDES 62

4-2.1. Xylomannans from the red seaweed Sebdenia polydactyla

4-2.2. Glucans from a commercial preparation

of rice bran (Oryza sativa)

4-3. ANALYTICAL METHODS

4-3.1. General

4-3.2. Sugar analysis 64

4-3.3. Sulfation 65

4-3.4. Sulfate estimation by Turbidometric method

4-3.5. Desulfation 66

4-3.6. Periodate oxidation and Smith degradation 67

4-3.7. Linkage analysis 67

4-3.8. Chromatography 4-3.8.1. Thin layer chromatography (TLC) 68

4-3.8.2. Size exclusion chromatography (SEC) 69

64

62

63

66

68

64

CHAPTER 4

62

4-3.8.3. Gas liquid chromatography (GLC) 70

4-3.8.4. Gas liquid chromatography –Mass Spectrometry (GLC-MS) 71

4-3.9. Spectroscopy 4-3.9.1. NMR Spectroscopy 71

4-3.9.2. UV-VIS spectroscopy 72

4-3.9.3. Infra-Red spectroscopy 72

4-3.9.4. Mass spectrometry 72

4-4. BIO – ASSAY

4-4.1. Cell culture and viruses 72

4-4.2. Anti viral assay 73

4-4.3. Virucidal assay 74

4-4.4. Cytotoxicity assay 74

4-4.5. Assay for anti coagulant activity 75

4-4.6. Influence of various treatment periods on the anti viral 75

activity of the compounds

REFERENCES 77

List of Publications

SUMMARY

99

98

71

72

Chapter 1

INTRODUCTION

Introduction: Chapter 1

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1-1. HERPES SIMPLEX VIRUS INFECTION

Herpes simplex virus (HSV) belongs to the Herpesviridae family which is a group of

large DNA viruses divided into three subfamilies of -, - and -herpes viruses. The human -

herpes viruses are HSV type 1 (HSV-1), HSV type 2 (HSV-2) and varicella zoster virus (VZV);

-herpes viruses include among other members human cytomegalovirus (CMV) and human

herpes viruses 6 and 7, while typical -herpes viruses are Epstein-Barr virus (EBV) and human

herpes virus 8.

The entry of HSV into host cells is a complex process initiated by specific interaction

between host cell-surface receptors and viral envelope glycoproteins (Ghosh, 2009a; Kleymann,

2005; Olofsson and Bergstrom, 2005; Schneider-Schaulies, 2000; Spear, 2004). All hitherto

investigated human herpes viruses, i.e. HSV-1, HSV-2, varicella-zoster virus (VZV),

cytomegalovirus (CMV), human herpes virus (HHV)-7 and HHV-8, except Epstein-Barr virus

(EBV) may utilize cell surface heparan sulfate (HS) chains for primary attachment (Akula, 2001;

Neyts, 1992; Secchiero, 1997; Trybala, 2002; WuDunn and Spear, 1989). Bergefall and co-

workers (2005) analyzed cell lines deficient in expression of glycosaminoglycans and postulated

that HSV-1 glycoprotein C (gC) binds to chondroitin sulfate (CS, characterized by E

disaccharide units) and that the CS-E unit is an essential component to function as a HSV-1

receptor. More recently this group showed that chondroitin 4-O-sulfotransferase-1 regulates the

E disaccharide expression of chondroitin sulfate required for HSV-1 infection (Uyama, 2006). In

case of HSV-1 and HSV-2, attachment to HS seems to be primarily mediated through

glycoprotein C (gC) although glycoprotein B (gB) may contribute to this function (Cheshenko

and Herold, 2002; Herold, 1991; 1994). Following HS binding, secondary entry receptors are


3 | P a g e

recognized in a cell-type specific manner, before virus entry requires the activity of another

glycoprotein (gD). gD can interact with the herpes virus entry mediator (HVEM, a member of

the tumor necrosis factor receptor family), with nectin-1 and nectin-2 (two related members of

the immunoglobulin super family) and, additionally, with specific sites in HS generated by

certain 3-O-sulfotranferases (Geraghty, 1998; Montgomery, 1996; O’Donnell, 2006; Spear and

Longnecker, 2003; Xu, 2005). The gD interactions possibly trigger conformational changes in

viral gB and gH/gL components thus initializing fusion between the viral lipid envelope and cell

plasma membrane (Perez-Romero, 2005; Spear, 2004). Subsequently, viral capsids and

teguments proteins are released into the cytoplasm of the host cell. Alternative pathways of

HSV-1 entry into the cell can occur in some cell-types through endocytosis. Endocytosed HSV-1

fuses with the endosomal membrane instead of the cytoplasmic membrane (Nicola, 2003). By

using a second entry pathway, viral glycoproteins can mediate HSV-1 entry via the apical

cellular surface in the form of a cell-to-cell spread, i.e., the movement of virons across the

narrow space between infected and adjacent uninfected cells (Cocchi, 2000; Kusche-Gullberg

and Kjellen, 2003).

1-2. THE RECEPTOR-BINDING SITE OF VIRAL GLYCOPROTEINS

HSV gC (designated gC1 and gC2 for HSV-1 and HSV-2, respectively) is a type 1

membrane glycoprotein that contains 511 (gC1) or 480 (gC2) amino acids and exhibits 65%

identity in the primary amino acid sequences between gC1 and gC2 (Swain, 1985). Although, at

present, the receptor-binding motifs of gC have not been completely elucidated, it is well

documented that gC1 has nine sites for N-linked oligosaccharides and numerous sites for O-


4 | P a g e

linked glycans. The latter are clustered at the N terminal part of the protein, which makes this

region structurally similar to mucins. The mucin-like region is not present in gC2. The eight

cysteins in gC1 form 4 disulfide bonds (Rux, 1996). Clusters of basic and hydrophobic amino

acids located between residues 129 and 160 of gC1 (Mardberg, 2001; 2002; Trybala, 1994) as

well as the mucin-like region (amino acids 33-123) of this protein (Tal-Singer, 1995) were

identified as important for HSV-1 attachment to cell surface HS/CS.

HSV-1 gB1 consists of 904 amino acids, and approximately 85% of the sequence is

homologous to its HSV-2 counterpart

(Heldwein, 2006). Most of the variability

between gB1 and gB2 is seen in a lysine-rich

region (amino acids 68-76), which is

responsible for binding to HS, but not

essential for the fusogenic activity of gB

(Laquerre, 1998). The crystal structure of gB1

has recently been revealed by Heldwein and

co-workers (2006). It occurs as a trimer with

each of the monomers divided in five

distinctive domains: I base, II middle, III core,

IV crown, and V arm (Fig. 1.1). Domain I

exhibited structural similarity to the pleckstrin

homology (PH) domain found in certain

phospholipid-binding cytoplasmic proteins.

Fig. 1.1. Ribbon diagram of a single gB1.

protomer.

Reprinted with permission from Heldwein E. E. et al., Science 2006, 313, 217-220 http://www.sciencemag.org. Copyright 2006, AAAS.


5 | P a g e

Specific hydrophobic/aromatic amino acids from this domain were found to be important for

fusogenic activity of gB suggesting that this protein is the potential fusogen (Hannah, 2007).

1-3. CELL-SURFACE GLYCOSAMINOGLYCAN (GAG) RECEPTORS

Heparan sulfate, an endogenous ligand for many viral proteins, is initially synthesized as

a copolymer of alternating (1→4)-linked β-D-glucuronic acid and N-acetylated α-D-glucosamine

and then undergoes various modifications in the Golgi apparatus (Lindahl, 1998). These

modifications include N-deacetylation and N-sulfation of glucosamine, C5 epimerization of

glucuronic acid to form iduronic acid residues, 2-O-sulfation of iduronic and glucuronic acid

residues, as well as 6-O-sulfation and 3-O-sulfation of glucosamine residue (Gorsi and Stringer,

2007). HS chains can provide specific binding sites for various proteins, exemplified by binding

of HSV-1 gB that depends on the presence of one or more 6-O-sulfate and 2-O-sulfate groups,

and gD that requires HS chain modified by 3-O-sulfotransferases isoforms II-VI (Chen, 2003;

O’Donnell, 2006; Shukla, 1999; Tiwari, 2005; Xia, 2002; Xu, 2005). A gD binding

octasaccharide motif (Liu, 2002) and a gC binding N-sulfated dodecasaccharide motif (Feyzi,

1997) were characterised confirming that HSV-1 utilizes a unique HS sequence for its entry. The

structural motifs of the glycoepitopes on HS chains are given in Fig 1.2. Recent results from cell-

based assays revealed that the inhibition of HSV-1 infection requires a unique sulfation moiety

(Copeland, 2008).


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1-4. ANTI VIRAL DRUGS: THE TREATMENT OF HERPES VIRAL

INFECTIONS

A successful anti viral agent must selectively target the virus particle or its replication

inside the cells without adverse effects on host cells. Since viruses are obligate intracellular

parasites which due to the absence of an own metabolism are strictly dependent on the cellular

nucleic acid- and protein-synthesizing machinery, the task to develop virus-selective anti viral

drugs has been difficult. In practice, every step in the infectious cell cycle of the virus i.e., its

attachment to cell surface molecules, its penetration into cells, nucleic acid synthesis, translation

Fig. 1.2. Receptor binding motif of cell surface heparan sulfate chains. The small letters and

numerals refer to sulfates binding to the nitrogen of glucosamine or to hydroxyl

oxygens of carbon atoms 2, 3 and 6 respectively. The chemical structures of

disaccharides critical for binding to gC and gD, respectively, are given. The

nomenclature for representation of glucosaminoglycans is as follows: GlcNAc = N-

acetylglucosamine; GlcNH2 = glucosamine; IdoA = L-iduronic acid; GlcA = D-glucoroic

acid; Xyl = xylose.

-(4GlcA1,4GlcNAc1)n---4IdoA1,4GlcNH21---(4GlcA1,4GlcNAc1)n---4IdoA1,4GlcNH21---4GlcA1,3Gal1-Gal1,4Xyl-Ser

3S

6S 2S 2S 6S

NS

H2N

O

OH

O

HOOC

O

O

OSO3

O3SO

-

-

-

O3SO

O

OH

O

HOOC

O

O

OSO3

OH

O3SO-

-

O3SHN-

Disaccharide relevant for gC binding

Disaccharide relevant for gD binding


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of viral proteins, virus assembly and egress from the cell or the free virions, can be a target for

anti viral drug. Some viral proteins (enzymes) or the virus replicative pathways, although similar

to those existing in cells, exhibit enough viral specificity to be selectively targeted by anti viral

agents. Most of the anti viral drugs currently approved for clinical (Fig 1.3) use are nucleoside

analogues targeting the viral DNA/RNA synthesis (Mercorelli, 2011; Vo, 2011).

Fig. 1.3. Anti HSV drugs licensed for clinical use.


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NH

N

NN

O

NH2

O

O

NH2

OH

O

N

N

O

NH2

OP

OH

OOH

OH

P OH

OH

O

O

OH

The breakthrough in anti viral treatment came in the late 1970s when the first specific

and selective anti viral drug, the nucleoside analogue acyclovir, was introduced for treatment of

HSV infections. Nowadays, the standard therapy for HSV infections is still acyclovir (Zovirax)

and related nucleoside analogues (Valtrex), famciclovir (Famvir) and penciclovir (Vectavir,

Denavir). Following uptake by the cell, acyclovir is selectively phosphorylated by the viral

(HSV, VZV or EBV) thymidine kinase (TK) and subsequently triphosphorylated by cellular

kinases. This selectivity renders acyclovir the low cytotoxicity since conversion to active forms

takes place in infected cells only, and this drug is also a better substrate for viral than cellular

DNA polymerase. Phosphorylated acyclovir acts by competition with cellular deoxyguanosine

triphosphate (dGTP) for binding to the viral DNA polymerase. Given the fact that acyclovir has a

higher affinity for viral DNA polymerase than its cellular competitor, this nucleoside analogue is

selectively incorporated into viral DNA where it terminates the DNA chain elongation.

Alterations in viral TK may lead to the generation of drug resistant variants of the virus (De

Clercq, 2001).

Valganciclovir Cidofovir

Fig. 1.4. Anti CMV drugs licensed for clinical use.

Foscarnet


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Nucleoside analogues can be used for episodic and/or long-time suppressive treatment of

HSV disease (Fig 1.4). Suppressive long-time therapy with valaciclovir is beneficial in that it

also reduces the host-to-host transmission of HSV including its asymptomatic shedding, a major

source of the virus spread in humans (Koelle and Wald, 2000; Sacks, 2004).

1-5. SULFATED POLYSACCHARIDES AS ANTI VIRAL AGENTS

The anti viral properties of sulfated polysaccharides have been known for almost 50 years

(Vaheri and Cantell, 1963; Vaheri, 1964a, Vaheri, 1964b). Accordingly, heparin (heparan sulfate

analogue) and several sulfated carbohydrate polymers including fucans (Adhikari, 2006;

Bandyopadhyay, 2011; Chattopadhyay, 2010; Hemmingson, 2006; Jiang, 2010; Karmakar, 2009;

2010; Mandal, 2007; Ponce, 2003; Rodriguez-Jasso, 2011; Saha, 2012; Sinha, 2010; Zhu, 2004),

carrageenans (Campo, 2009; Carlucci, 1997; Silva, 2010; Tischer, 2006) sulfated galactans

(Bandyopadhyay, 2011; Chattopadhyay, 2007; 2008; Mazumder, 2002; Matsuhiro, 2005; Salehi,

2011; Zibetti, 2009), pentosan polysulfate (Baba, 1988; Viana, 2011), cellulose sulfate

(Anderson, 2002; Christensen, 2001), dextran sulfate (Witvrouw and De Clercq, 1997),

xylogalactans (Damonte, 1996), xylomannan sulfate (Ghosh, 2009b; Mandal, 2008; Recalde,

2012), mannans (Fernandez, 2012), spirulan (Lee, 2001; 2007; Rechter, 2006) and others (for

review see Ghosh, 2009a; Pujol, 2007) have been found to inhibit HSV because of their

structural similarity to HS and therefore are potential anti HSV drug candidates (Balzarini and

Van Damme, 2007; Costa, 2010; Ghosh, 2009a; Pujol, 2007; Wijesekara, 2011; Witvrouw and

De Clercq, 1997). Sulfated polysaccharides act as anti viral agents in cell culture because these

charged polymers mimic HS chains on cell-surface proteoglycans and block viral attachment by

competitive inhibition. A novel approach to inhibiting HSV-1 infection by targeting the gD


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mediated membrane fusion step has recently been described (Copeland, 2008). This inhibition

was achieved by using a unique 3-O-sulfated octasaccharide, which was generated from heparin

using an enzymatic approach. The results of this study also demonstrate that the inhibition of

HSV-1 can be blocked by saturating the viral envelope glycoprotein gD using a small molecule

of defined structure. The anti herpetic properties of sulfated polysaccharides may depend not

only on their charge density but also on the characteristics of their uncharged portions which

may be involved in hydrophobic and hydrogen bonding interactions. Mardberg and co-workers

(2001) reported that hydrophobic interactions, in addition to electrostatic forces, are decisive for

the CS as well as HS binding to viral glycoprotein gC. It has been suggested that sulfated

carbohydrates can elicit profound conformational changes in hydrophobic regions of proteins

and cause disassembly of complex proteins consisting of subunits (Sedlak and Antalik, 1998).

Therefore, the interaction of the methyl groups of fucoidan with the hydrophobic pocket of HSV-

1 gC seems to be important in the binding of the polysaccharide to the viral glycoprotein. While

sulfated oligosaccharide chains can bind to and block the viral attachment/receptor-binding

proteins, it was speculated that for this class of viral inhibitors the hydrophobic group might

additionally insert into the viral lipid envelope (Uryu, 1992). This might lead to a destabilization

and irreversible inactivation of the virion.

Finally, in addition to the polysaccharide-mediated anti viral effects directed to the

cell surface (viral receptor binding, entry, fusion), a second type of effects may play a role, i.e.

the induction of intracellular events contributing to the anti viral activity of sulfated

polysaccharides. As the binding of a number of known polysaccharides to cell surface receptors

can induce intracellular signaling pathways, this second type of effects should be additionally

taken into consideration. As an example, the anti cytomegaloviral effect of spirulan-like


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polysaccharides was demonstrated to be composed of these two anti viral activities, i.e. an

inhibition of HCMV entry on the one side in addition to the induction of intracellular anti

HCMV effects on the other side (Rechter, 2006). Due to the fact that the replication efficiency of

most viruses is dependent on specific intracellular signaling pathways, the inhibition or the

induction of particular signaling of surface-binding polysaccharides can provide a significant

part of the overall anti viral activity. One explanation for such intracellularly produced activity is

the stimulatory effect of sulfated polysaccharides onto interferon production with the

consequence of a broad anti viral effect.

Recent structural studies showed that sulfated polysaccharides from marine algae represent

a group of macromolecules possessing more properties in common than previously thought.

These macromolecules have anti HSV properties, particularly due to their ability to imitate

patterns of sulfate substitution on gags present in cell membranes. Because the structures of algal

sulfated polysaccharides are complicated and as many studies of anti viral activity were carried

out using relatively crude polysaccharide preparations, it is presently not easy to determine the

overall relationship between activity and structure. However, the results of many studies

demonstrate that the antiviral activity of sulfated polysaccharides is not merely a function of high

charge density and chain length but also distinct structural characteristics. Recent studies also

demonstrated that sulfated polysaccharides could be used as a vaginal antiviral formulation

without disturbing essential functions of the vaginal epithelial cells and normal bacterial flora.

But, the complexity and heterogeneity of sulfated polysaccharides, and their anti HSV activities

are not attractive within current models of drug development. The combination of structural

elucidation and assignment of antiviral activity to specific structural features can only improve the

potential of sulfated polysaccharides as anti HSV agents.


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To date, the performance of these macromolecules in efficacy trials has been disappointing

(Cohen, 2008; Grant, 2008), but next-generation concepts now in or approaching clinical trials

offer improved prospects for efficacy (Klasse, 2008). The most plausible approach involves a

combination of several drugs, preferentially targeting different steps in the viral infection

process. Because sulfated polysaccharides are safe and acceptable (Bollen, 2008; Kilmarx,

2008), development of several second-generation combination formulation based on first

generation lead candidates may be more effective (Bandyopdhyay, 2011; Brache, 2007; Ghosh,

2009b; Karmakar, 2010; Liu, 2002; Mandal, 2008; 2010; Saha, 2010; Sinha, 2010). Marine algae

are an important source of sulfated polysaccharide. A recent project completed in our laboratory

helped to develop a library of sulfated polysaccharides having broad spectrum antiviral activities

from selected Indian marine algae and their potencies are different. It is, therefore, probable that

sulfated polysaccharides from other marine algae may have superior efficacy. This probability

warrants further study involving isolation, purification, chemical characterization and antiviral

potency of sulfated polysaccharides from other marine algal species. Polysaccharides from

higher plants on the other hand do not possess sulfate group, but it is possible to introduce sulfate

group by chemical means. Structurally defined polysaccharides generated by chemical sulfation

may also produce drug candidate with higher potency.

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AIM OF THE THESIS

The major goal of this research is to develop anti viral drug candidates from Indian

marine red alga Sebdenia polydactyla and a commercial preparation of rice bran (Oryza

sativa).

The strategy adopted to achieve this goal involves:

Isolation / generation of the anti viral drug candidates i.e., anionic polysaccharides from

the two different sources.

Comparison of anti viral activity of the sulfated polysaccharides with standard viral

inhibitor such as heparin and dextran sulfate.

Purification of the crude extract and screening of anti viral activity of the pure compound.

Chemical modification of the active principles.

Investigation on the molecular basis for their anti viral potency.

Finally, determination of the structural features of the anti viral drug candidates.

Results

and

Discussions

Chapter 2

STRUCTURAL FEATURES AND ANTI VIRAL ACTIVITIES OF SULFATED

XYLOMANNANS FROM THE RED SEAWEED

Sebdenia polydactyla

Chemical and anti viral properties of sulfated xylomannans: Chapter 2

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2-1. INTRODUCTION

Algae have been repeatedly recognized as producers of biologically active

substances. Specific studies on seaweeds, carried out in the Atlantic, Pacific and Indian

oceans, have demonstrated anti bacterial, anti fungal and anti viral (Cumashi, 2007;

Damonte, 2004; Ghosh, 2009a; Lahaye, 1991; Mayer, 2009a, 2009b; Renn, 1992; Smit,

2004) activities. Seaweeds grow abundantly along Indian coastal line and represent an

important biomass poorly exploited (Wealth of India, 1985).

In recent years, many compounds having potent antiviral activity in cell cultures

have been detected and some of these compounds are currently undergoing either

preclinical or clinical evaluation. Among these antiviral substances, sulfated

polysaccharides from marine algae and of synthetic origin are noteworthy. These

polysaccharides include mainly agarans, carrageenans, fucans, mannans, rhamnans, and

ulvans (Arad, 2006; Damonte, 2004; Witvrouw and De Clercq, 1997). Indeed, some of

these macromolecules are in various phases of clinical trials as microbicides (Balzarini,

2007; Kilmarx, 2006; Nikolic, 2007; Kleymann, 2005; Pujol, 2007; Ghosh, 2009a). Thus,

evaluating the potential of sulfated polysaccharide extracted from marine algae as anti

herpes simplex virus (HSV) drug candidates will be of considerable interest.

One of the important parameter for the antiviral activity of a polysaccharide is its

degree of sulfation (DS) i.e., the number of sulfate groups per monosaccharide residue. In

addition, the antiviral potency has been thought to be dependent on molecular mass

(Witvrouw and De Clercq, 1997; Pujol, 2007; Ghosh, 2009a). This chapter describes

isolation, purification, structural features and activity of sulfated polysaccharides present

in red seaweed Sebdenia polydactyla against Herpes Simplex virus. The possibility to


16 | P a g e

generate xylomannan derivatives by chemical sulfation in the O-positions along the

polysaccharide has led to the synthesis of various sulfated derivatives with different

degrees of sulfation. With these tailored modifications a range of compounds have been

generated that have potential anti HSV activity and low cytotoxicity.

2-2. CHEMICAL CHARACTERIZATION OF SULFATED


2-2.1. Preparation of depigmented algal power and sugar composition

analysis

The central goal of this study is to determine the structural features of

polysaccharides from the marine red alga S. polydactyla grown along the coast of Okha,

India. The collected seaweeds were washed thoroughly with tap water, dried by forced air

circulation, pulverized in a blender and the algal powder was depigmented using

Fig. 2.1. Scheme for the preparation of depigmented algal powder from the red seaweed Sebdenia polydactyla

Depigmentation

DAP

Sebdenia polydactyla (Sp) Algal powder


17 | P a g e

sequential extraction with petroleum ether and acetone as solvent in a Soxhlet apparatus

to yield depigmented algal powder (DAP) as shown in Fig 2.1.Therefore, first step should

obviously be the determination of sugar composition of the algal powder and, on the

basis of the information obtained, to devise a strategy for the extraction of

polysaccharides. GLC-MS analysis of the alditol acetates derived from the depigmented

algal powder suggests that it contained a number of sugars including large quantities of

xylose and mannose (Fig 2.2). For the determination of monosaccharide composition,

A

min

min

m/z

m/z

m/z m/z

Gal Glc Man Inositol (Internal standard)

Xyl Fuc

Fuc Rha

50 100 150 200 250 3000

25000

50000

75000

100000 43

115 170129

9969 85 157145 187 231

73289201 21741

303

50 75 100 125 150 175 200 2250e3

50e3

100e3

43

115

145103

85217127 18715873

20017555 218

50 100 150 200 250 300 3500

25000

50000

43

115

139

103 187 217157170 25973 28941 361

Ara

B

Fig. 2.2. GLC-MS analysis of the alditol acetates derived from (A) standard

sugars and (B) depigmented algal powder (DAP)

Fig. 2.2. The GLC-MS analysis of the alditol acetates derived from standard sugars.

Xylo

se

Xylo

se

Man

nose

Man

nose

G

alac

tose

Gal

acto

se

Glu

cose

Glu

cose

In

osi

tol

Inosi

tol

(Int.

Std

)

min


18 | P a g e

the DAP was hydrolyzed in 2M H2SO4, the sugars released were reduced and acetylated

as described in section 4-3.2. The derived alditol acetates were separated by GLC and

identified by their (i) retention time relative to that of myo inositol hexa-acetate (internal

standard) and (ii) mass spectral fragmentation patterns. The presence of xylose and

mannose indicates the presence of xylan and/or mannan and/or xylomannan.

2-2.2. Isolation and sugar composition of polysaccharide fractions

The strategy for the extraction of polysaccharides is given in Fig 2.3.

Here, the depigmented algal powder (DAP) was extracted with water. Water is a cheap

nontoxic and polar solvent and therefore most suitable for the extraction of

F1D-Sm

F1D-Sm

Fig. 2.3. Scheme for the isolation of polysaccharides from the red seaweed S. polydactyla, their purification and chemical modification.

Figure. 2.4. Scheme for the isolation of polysaccharides from the red seaweed S. polydactyla, their purification and chemical modification.

Depigmentation

Depigmentation

Extraction with Water

Extraction with Water

Desulfation

Desulfation

Desulfated polymer (SpWED)

Desulfated polymer

(SpWED)

Water extracted polymer (SpWE)

Water extracted

polymer (SpWE)

Desulfation

Desulfation

F1D

F1D Smith

degradation

Smith

degradation

Size exclusion chromatography


DAP

DAP

Sebdenia polydactyla (Sp) Sebdenia polydactyla (Sp)

Insoluble (INS)

Insoluble (INS)

F1


Sulfation using SO3-

pyridine complex, 30°C

0.5 h 1.0 h 1.5 h 2.0 h

F1S1 F1S3 F1S2 F1S4

Reaction time:


19 | P a g e

polysaccharides which contained a number of hydroxyl and other polar groups such as

sulfate. Isolation having been achieved the step that follows is purification. The water-

extracted material was purified by repeated precipitation with dehydrated ethanol (4

vols.). Two fractions were isolated based on their solubility in 80% ethanol: SpWE-S, the

aqueous 80% ethanol soluble polymeric fraction and SpWE, ethanol insoluble fraction.

The SpWE-S fraction contained galactose as the major constituent sugar (Fig 2.4). The

water extracted (SpWE), which amounted to 14% of DAP dry weight, contained mannose

Fig. 2.4. GLC-MS analysis of the alditol acetate derived from the (A) water

soluble fraction (SpWE); (B) ethanol soluble fraction (SpWE-S).

A

(c)

50 100 150 200 2500e3

50e3

100e3

43

115

145103

85217127 18715873 20017555 289218

min

min

min

min

m/z

m/z

m/z 50 100 150 200 250 300 3500

25000

50000

43

115

139

103 187 217157170 25973 28941 361

Xylo

se

Man

nose

Gal

acto

se

Inosi

tol

Xylo

se

Inosi

tol

B


20 | P a g e

and xylose as the glycosyl residues (Table 2.1; Fig 2.4). This fraction did not contain any

galactose residue. Neither any methylated sugars were detected during GLC- MS analysis

of the derived alditol acetates.

2-2.3. Purification of the sulfated xylomannans by chromatography The fraction SpWE was purified by size exclusion chromatography on a

Sephacryl S-300 column using 0.5 M sodium acetate buffer (pH 5.0) as eluant. Two

peaks were obtained (Fig 2.5). Sub fractions, which appeared at Kav 0.26 to to 0.61, were

mixed and the recovered material has been named as F1 fraction. It accounted for 78% of

the total sugar eluted from the column. This fraction eluted as a symmetrical peak by re-

chromatography. Sugar compositional analysis suggests that F1 contained mannose and a

small amount of xylose (Table 2.1). Therefore this fraction is a xylomannan. Fraction F2,

also contained xylose and mannose as constituent sugars and hence, is a xylomannan.

Sugar Composition SpWE F1 F2 F1D F1S1 F1S2 F1S3 F1S4

Total Sugar, %a 42 46 39 67 43 42 40 38

Degree of Sulfationb 0.5 0.6 0.5 Nd 1.0 1.2 1.5 1.6

Xylose, %c 49 31 28 30 32 31 26 25

Mannose, %c 51 69 72 70 68 69 74 75

Table 2.1. Sugar composition of fractions generated from Sebdenia polydactyla

The sugar composition (molecular %) was determined for the following fractions: water-

extracted xylomannan (SpWE), purified major xylomannan sulfate (F1), sulfated derivatives of

xylomannan obtained at various time points in the sulfation process (F1S1, F1S2, F1S3 and F1S4

at 0.5, 1.0, 1.5 and 2.0 h respectively), the minor xylomannan fraction (F2) and desulfated

xylomannan (F1D). aPercent weight of fraction dry weight. bNumber of sulfate groups per

monosaccharide residue. cMolecular percentage of neutral sugar. Nd, not determined.


21 | P a g e

The xylomannan containing fraction (F1) contained 8% (w/w) sulfate and

subjected to further structural analysis. This polymer had a positive specific rotation

[ ]D30

+42 o (c 0.2, H2O) and is soluble in water.

2-2.4. Desulfation and further sulfation

It is well known that the bulky and negatively charged sulfate group dictates the

physical and chemical properties of anionic polysaccharide solution. Therefore, it is

highly probable that this group will also dictate their biological properties. Indeed, it has

been shown that for a particular class of polysaccharide, the higher the charge density, the

higher is its anti viral activity (Ghosh, 2009a). To investigate the effect of sulfate group

we have desulfated the purified (F1) sulfated xylomannan (as pyridinium salt) by

solvolytic desulfation (Falshaw and Furneaux, 1998) to produce a desulfated derivative

(F1D). Preliminary experiments (data not shown) showed a higher recovery with this

method compared to methanol-HCl and auto-desulfation methods of Percival and Wold,

Fig. 2.5. Purification of the crude water extracted fraction (SpWE) from S. polydactyla by SEC on S-300 column.

Kav.

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.00 0.19 0.39 0.58 0.77 0.97 1.00 1.00 1.00

(F1)

(F2)

Res

po

nse

fac

tor


22 | P a g e

1963. Notably, the sugar composition of F1 and its desulfated derivative (F1D) were

comparable (Table 2.1).

At the same time, in separate experiments, the purified xylomannan sulfate was

further sulfated under various conditions as described in Materials and Methods (Section

4-3.3) to yield derivatives F1S1, F1S2, F1S3 and F1S4 with various degrees of sulfation

(Table 2.1)

2-2.5. Infra red (IR) spectroscopy

IR spectroscopy is an important tool for the characterization of sulfate group. The

fourier transform (FT)-IR spectrum of SpWE (Fig 2.6) as well as its purified fraction F1

showed an intense absorption band in

the region 1252 cm-1

related to S=O

stretching vibration of the sulfate

group (Hirst, 1965; Lloyd, 1961;

Turvey and Williams, 1962). An

additional sulfate absorption band at

830 cm-1

(C-O-S, secondary equatorial

sulfate) indicated that the sulfate group

is located at equatorial position i.e., at

C-4 or C-3 of the mannopyranose residue (Rees, 1963). On solvolytic desulfation of the

xylomannan (F1), a desulfated derivative is obtained as described earlier and these

absorbances are greatly reduced demonstrating that they were associated with sulfate

groups (Fig 2.7).

Fig. 2.6. FT-IR spectrum of water soluble purified

polysaccharide fraction (F1) obtained from

Sebdenia polydactyla.

830 cm-1

830 cm-1

11

30

15

20

25

4000 600100020003000

%T

Wavenumber [cm-1]

Sulfate band (1252 cm-1)


23 | P a g e

The FT-IR spectra of the further sulfated xylomannans (F1S1 and F1S3) the band at 1252

cm−1

assigned to the >S=O stretching vibration of the sulfate group is also clearly visible

(Fig 2.8).

2-2.6. Molecular mass

Another important parameter is

molecular weight which contributes to anti

viral activity of the polysaccharides isolated

from marine algae. For a family of

polysaccharides, the higher the average

molecular mass, the higher is the anti viral

activity in many cases. Therefore, to

investigate the role of molecular mass in the

Fig. 2.7. FT-IR spectrum of the desulfated xylo-

mannan fraction (F1D) obtained from

red seaweed Sebdenia polydactyla.

30

110

40

60

80

100

4000 600100020003000

%T

Wavenumber [cm-1]

Sulfate band 1252 cm-1

4000

4000

3500

3500

3000

3000

2500

2500

2000

2000

1500

1500

1000

1000

500

50

60

70

80

90

100

110

2

% T

3

1

Sulf

ate

ban

d

~1

252

cm

-1

Wave number, cm-1

Fig. 2.8. FT-IR spectra of (1) desulfated (F1D) and (2 and

3) further sulfated (F1S1 and F1S3) xylomannan. A series of xylomannans with different degree of sulfation were prepared by further sulfation of the purified xylomannan sulfate (F1).

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0 0.05 0.14 0.24 0.33 0.43 0.52 0.62 0.71 0.81 0.90 1.00

Kav.

Fig. 2.9. SEC elution profile of the fraction

F1 on Sephacryl S-300 column.

Res

po

nse

fac

tor

Sulfated

Xylomannan

(F1)


24 | P a g e

antiviral activity of sulfated xylomannans we have tried to determine their molecular

mass by size exclusion chromatography (SEC). SEC of F1 on Sephacryl S-300 column

suggests that the polymer is homogeneous (Fig 2.9). Based on calibration with standard

dextrans, the apparent molecular mass of the polysaccharide would be 150 kDa.

2-2.7. Smith degradation

To get more information on the structure of xylomannans, periodic acid

oxidation of F1D was carried out (Fry, 1988). The oxidized polymer was reduced with

sodium borohydride and subjected to mild acid hydrolysis according to the usual Smith

degradation conditions. After separation from low-molecular-mass fragments, a Smith

degraded material (F1D-Sm) containing mannose as the only component sugar was

obtained in 50% yield.

2-2.8. Glycosidic linkage analysis

Methylation analysis of the desulfated macromolecule (F1D) gave, inter alia,

1,3,5-tri-O-acetyl-2,4,6-tri-O-methylmannitol residue suggesting that the xylomannan

sulfate has (1 3)-linked backbone (Fig 2.10). The presence of large proportion of

terminal xylopyranosyl and 1,3,5,6-tetra-O-acetyl-2,4-di-O-methylmannitol residues

suggest that the polymer is branched at position 6 (Table 2.2). Linkage analysis of the

purified xylomannan sulfate (F1) yielded a variety of mono-, di- and trimethylated

products indicating that the structure of this polymer is highly complex.


25 | P a g e

The increase in the proportions of 1,3,5-tri-O-acetyl-2,4,6-tri-O-methylmannitol

after desulfation, as a consequence of decreased proportions of 1,2,3,5,6-penta-O-acetyl-

4-O-methyl- and 1,3,5,6-tetra-O-acetyl-2,4-di-O-methylmannitol residues, suggests that

sulfate esters, when present, are located at position 6 and 2 of the (1 3)-linked

mannopyranosyl residues. Small amounts of (1 6)-linked manno-pyranosyl residues

were also present.

The results obtained from the methylation analysis of the Smith degraded

polymer (F1D-Sm) showed (Table 2.2) the degraded polysaccharide to be a linear

mannan built up of (1 3)-linked mannopyranosyl residues.

min

Fig. 2.10. Total ion chromatogram of partially methylated alditol acetates (PMAA)

derived from (A) the desulfated xylomannan (F1D) and (B) its Smith

degraded derivative (F1D-Sm). 1: 1,5-di-O-acetyl-2,3,4-tri-O-methylxylitol;

2: 1,5,6-tri-O-acetyl-2,3,4-tri-O-methylmannitol; 3: 1,3,5-tri-O-acetyl-2,4,6-

tri-O-methylmannitol; 4: 1,3,5,6-tetra-O-acetyl-2,4-di-O-methylmannitol;

5: 1,2,3,5,6-penta-O-acetyl-4-O-methylmannitol; 6: hexa-O-acetyl-myo-

inositol; X: impurity.

min

B

Х

A

1 2

3

4 5

6

Х Х

Х


26 | P a g e

The disappearance of 1,5-di-O-acetyl-2,3,4-tri-O-methylxylitol, and 1,3,5,6-tetra-

O-acetyl-2,4-di-O-methylmannitol residues suggest the presence of single stub of xylose

residues at 6 position of the mannose units of native xylomannan sulfate.

Notably, the partially methylated alditol acetates (PMAA) were separated by GLC

and identified by their (i) retention time relative to that of inositol hexa-acetate (internal

standard) and (ii) mass spectral fragmentation patterns. Mass spectral (EI) fragmentation

pattern of 1,5-di-O-acetyl-2,3,4-tri-O-methylxylitol, 1,5,6-tri-O-acetyl-2,3,4-tri-O-

methylmannitol, 1,3,5-tri-O-acetyl-2,4,6-tri-O-methylmannitol, 1,3,5,6-tetra-O-acetyl-

2,4-di-O-methylmannitol, 1,2,3,5,6-penta-O-acetyl-4-O-methylmannitol and myo-

inositol hexaacetate are given below (Fig 2.11).

Methylation products

m/z values

Peak areab

F1 F1D F1D-Sm

2,4,6-Manpa 43, 45, 101, 118, 129, 161 and 234 13 47 100

2,3,4-Manp 43, 118, 129, 189 and 233 4 2 Nd

2,4-Manp 43, 118, 129, 189 and 234 34 26 Nd

4-Manp 43, 129, 189, 202 and 262 22 1 Nd

Manp 43,….103, 115, 128, 145, 157, 171

187, 218, 260 and 290

6 Nd Nd

2,3,4-Xylp 43, 101, 102, 117, 118, 161 and 162 21 24 Nd

Table 2.2. Partially methylated alditol acetates derived from sulfated xylomannan (F1),

its desulfated (F1D) and desulfated Smith degraded (F1D-Sm) derivative.

a2,4,6-Manp denotes 1,3,5-tri-O-acetyl-2,4,6-tri-O-methylmannitol, etc. bPercentage of

total area of the identified peaks. Nd, not determined.


27 | P a g e

m/z

m/z

m/z

m/z

m/z

m/z

50 75 100 125 1500e3

10e3

20e3

30e3

40e3

43

101

11888

7359

161129

50 75 100 125 150 175 200 2250

5000

10000

15000

43

118

87 129

10174

59

18941

234152

50 75 100 125 150 175 200 225

0

25000

50000

75000

100000 43

118

101129

74

59 87161

234202143 217174

161

118

234

174 101

129

45

CHOAc

CHDOAc

CHOMe

CH2OMe

CHOMe

CHOAc

189

118

234 129

CHOAc

CHDOAc

CHOMe

CH2OAc

CHOMe

CHOAc

CH2OAc

CHDOAc

CHOMe

CHOMe

CHOMe

161

118

117

162

101

102

(1)

(2)

(3)

(4)

50 75 100 125 150 175 200 2250e3

100e3

200e3 43

11887

129

102

18959 71 162146 220 233

m/z

m/z

162

118

233 102

129

C H O A c

C H D O A c

C H O M e

C H 2 OAc

C H O M e

C H OMe


28 | P a g e

2-2.9. Nuclear magnetic resonance (NMR) Spectroscopy

NMR spectroscopy is a convenient method that gives valuable information on

polysaccharide structures (Chattopadhyay, 2007; Ghosh, 2009b; Pereira, 1999; Ray,

2006). We employed NMR analysis to determine the anomeric configuration and the

overall structure of the sulfated xylomannan. The 1H NMR spectrum of the purified

sulfated xylomannan (F1; Fig 2.12) shows the presence of atleast two broad signals in the

anomeric region.

Fig. 2.11. EI-MS fragmentation pattern of partially methylated alditol acetates

derived from the desulfated xylomannan (F1D) of Sebdenia polydactyla.

(1) 1,5-di-O-acetyl-2,3,4-tri-O-methylxylitol; (2) 1,5,6-tri-O-acetyl-2,3,4-

tri-O-methylmannitol; (3) 1,3,5-tri-O-acetyl-2,4,6-tri-O-methylmannitol;

(4) 1,3,5,6-tetra-O-acetyl-2,4-di-O-methylmannitol; (5) 1,2,3,5,6-penta-O-

acetyl-4-O-methylmannitol; (6) myo- inositol hexaacetate.

m/z

m/z

50 75 100 125 150 175 200 225 250

0

5000

10000

15000

20000

2500043

129

87

74

100 18911659

262202

189

262

202

129

CHOAc

CHDOAc

CH2OAc

CHOMe

CHOAc

CHOAc

(5)

50 75 100 125 150 175 200 225 250 2750

25000

5000043

168

126

115

210

1579773 1991398158 270186 241228

(6)

m/z

m/z


29 | P a g e

The desulfated xylomannan (F1D) also

showed two anomeric resonances, one

at 5.119 ppm and the other at 4.426 ppm

(Fig 2.12). The anomeric configuration

of mannose and xylose residues of a

structurally related sulfated

xylomannans from Nothogenia

fastigiata have been reported to be -

and -, respectively (Kolender, 1995;

1997; Mandal, 2008; Matulewicz and

Cerezo, 1987). Therefore, signals at

5.119 and 4.426 ppm were tentatively

assigned to anomeric protons of -

(1→3)-linked mannopyranosyl and -

linked terminal xylose residues,

H1

α-M

an

p

H1

α-M

an

p

H1

β-X

yl p

B

C

Х

Х

HOD

HOD

Chemical shift

Fig. 2.12. 1H NMR spectra of (A) the purified sulfated

xylomannan (F1) (B) the desulfated xylo-

mannan (F1D) and (c) its Smith degraded

degraded derivative (F1D-Sm). The spectrum

for samples was recorded in D2O solution.

HOD, signal for deuterated water; X, Impurity.

ppm

HO

D

H1

of

-Xy

l p

H1

of

α-M

an

p

Ring Protons A

ppm

Chemical shift


30 | P a g e

respectively. The disappearance of the later signal in the NMR spectrum of the Smith-

degraded polymer (F1D-Sm) confirms the presence of β-linked terminal xylose (Fig

2.12). Like desulfated xylomannan (F1D), this degraded material (F1D-Sm) also include

resonances characteristic of polysaccharides such as signals from ring protons (H2 to H6)

between 3.2 and 4.2 ppm. The disappearance of the signals at 3.307, 3.441 and 3.631

ppm in the

1H NMR spectrum of the Smith degraded polymer (F1D-Sm) suggests that

they originated from the ring protons of xylose residues. Therefore, NMR analysis

confirmed the results of methylation analysis and indicated the presence of -linked

mannopyranosyl and -linked terminal xylopyranosyl residues.

2-3. BIOLOGICAL ACTIVITIES OF SULFATED XYLOMANNANS

FROM Sebdenia polydactyla

2-3.1. Cytotoxicity.

The crude water extract from S. polydactyla (SpWE), the major purified sulfated

xylomannan (F1) and its desulfated derivative (F1D) were initially evaluated for

cytotoxicity by assessing their effects on Vero cell viability. For comparative purposes,

heparin was simultaneously assayed as known reference polysaccharide. No effect on cell

viability was observed with any of these compounds at concentrations up to 1,000 μg/ml.


31 | P a g e

2-3.2. Anti herpetic activities.

The results of cytotoxicity and antiviral activities of purified sulfated xylomannan (F1)

from S. polydactyla, its desulfated fraction (F1D) and the four further sulfated derivatives

(F1S1 to F1S4) as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium bromide (MTT) and plaque reduction assays, respectively are summarized in

Table 2.3. The purified polysulfate F1 may be considered as a good inhibitor of HSV-1,

strain F, with an IC50 value of 2.8 µg/ml. On the other hand, the desulfated derivative

F1D was inactive against this virus up to a concentration of 10 µg/ml.

Compound

CC50a, µg/ml IC50

b, µg/ml SI (CC50/ IC50)

Selectivity indices

F1

1000 2.8 0.5 357

F1S1

1000 0.7 0.1 1428

F1S2

1000 0.6 0.2 1666

F1S3

1000 0.47 0.05 2127

F1S4

1000 0.35 0.03 2857

F1D

1000 10 Inactive

Heparin 1000 1.2 0.3 833

Dextran sulfate

8000 1000 2.1 0.4 476

Table 2.3. Anti viral activities and cytotoxicities and selectivity indices of sulfated

xylomannan fractions from Sebdenia polydactyla.

Compound

CC50a, µg/ml IC50

b, µg/ml SI

c (CC50/ IC50)

F1

1000 2.8 0.5 357

F1S1

1000 0.7 0.1 1428

F1S2

1000 0.6 0.2 1666

F1S3

1000 0.47 0.05 2127

F1S4

1000 0.35 0.03 2857

F1D

1000 10 Inactive

Heparin 1000 1.2 0.3 833

Dextran sulfate

8000 1000 2.1 0.4 476

Table 2.3. Anti-viral and cytotoxic activities of sulfated xylomannan fractions from S.polydactyla

The fractions tested were purified xylomannan sulfate (F1), sulfated derivatives of xylomannan

obtained at different time points in the sulfation process (F1S1, F1S2, F1S3 and F1S4 at 0.5, 1.0,

1.5 and 2.0 h, respectively) and desulfated xylomannan (F1D) aCC50 (Cytotoxic concentration 50%): Concentration required to reduce the number of viable

Vero cells by 50% after 48 h of incubation with the compounds. This concentration was ≥ 1000

µg/ml for all the compounds. bIC50 (Inhibitory concentration 50%): Concentration required to reduce plaque number in Vero

cells by 50 %. Each value is the mean of two determinations ± SD.


32 | P a g e

The conclusion that can be drawn is that the anti viral activity of this xylomannan

is linked to the anionic features of the molecule, given mainly by the presence of sulfate

groups. To further assess this feature, the four further sulfated compounds obtained by

chemical modification of the purified polysaccharide F1 were also tested against HSV-1

(Fig 2.13). In all cases, the further sulfated derivatives exhibited a more potent antiviral

activity in comparison to the native xylomannan F1, with values of IC50 ranging from

0.35 to 0.7 µg/ml. The small differences observed in the IC50 values of the further

sulfated compounds were related to the sulfate content, with higher antiviral activity

associated to a greater degree of sulfation (DS). Moreover, the fractions F1S1 to F1S4,

which presented DS values in the range 1.0 to 1.6, showed a significant increase in their

antiviral potential in comparison to the native F1, with a DS of 0.6 (Table 2.1). These

data allow to confirm the importance of this parameter in the inhibitory activity of

sulfated polysaccharides. Furthermore, the four further sulfated fractions exhibited high

Fig. 2.13. The IC50 values of different sulfated polysaccharides against HSV. The

purified sulfated xylomannan (F1) were obtained from the red seaweed S.

polydactyla. F1D is the desulfated derivative of F1. Heparin and Dextran

sulfate 8000 are commercial standard sulfated polysaccharides with anti

HSV activity.

D

ex

tra

n S

ulf

ate

80

00

F1 He

pa

rin

F1D

IC50 (

µg

/ml)


33 | P a g e

selectivity indices ( 1428 to 2857) due to their lack of toxicity for Vero cells. No

effect on cell viability was observed with any of these compounds at concentrations up to

1000 µg/ml. They also showed a higher inhibitory effect and selectivity when compared

with other well known sulfated polysaccharides such as heparin and dextran sulfate 8000,

assayed as reference substances.

The major constituent is a sulfated xylomannan (F1) with complex structure and

a molecular mass of 150 kDa. Recent studies showed that sulfated xylomannan from S.

hatei and N. fastigiata contained a backbone of -(1 3)-linked D-mannopyranosyl

residues substituted at different positions with single stub of -D-xylopyranosyl residues

and sulfate groups (Kolender, 1995; 1997; Mandal, 2008; Matulewicz, 1987). The

polysaccharide of the present study has a similar backbone, but it differed from N.

fastigiata xylomannan in the position of xylopyranosyl residues and from S. hatei

polymer in the location of sulfate group. Moreover, it is highly branched, an average of

25 branching points being present in every hundred mannopyranosyl residues. Therefore,

the structure of the polysaccharide described in the present paper is different from these

known sulfated xylomannans and it is obvious that its activity against HSV will be

different. Furthermore, the native sulfated xylomannan was desulfated and further

sulfated using SO3 =

pyridine to yield a series of polysaccharides with different sulfate

content (DS values varies from ~0.5 to 1.6).

Sulfated polysaccharides are known to affect the growth of animal viruses including

HSV (Kleymann, 2005; Pujol, 2007; McReynolds and Garvey-Hague, 2007). Herold and

coworkers (1996) showed that N-sulfation and the presence of carboxyl groups on

heparin are key determinants for HSV interactions with host cells, since N-desulfation


34 | P a g e

and carboxyl reduction abolishes heparin’s antiviral activity. The sulfated xylomannan

from S. polydactyla drastically lost activity when desulfated. Furthermore, this

macromolecule (F1) and its further sulfated derivatives (F1S1 to F1S4) exhibited potent

inhibitory effect on HSV infection. As can be seen in Tables 2.1 and 2.3, the level of

antiviral efficacy as determined by IC50 and SI values, was directly related to the content

of sulfate groups in the molecule. However, the four further sulfated samples, exhibiting

a variation of DS from 1.0 to 1.6, were highly inhibitory of HSV-1 multiplication. Thus,

it appears that a content of one sulfate group per monosaccharide unit (DS of 1.0) is

enough to guarantee a good level of selective antiviral effect.

It is believed that the in vitro efficacy of sulfated polysaccharides is mainly because

of their ability to inhibit the attachment of the virion to the host cell surface (Kleymann,

2005; Pujol, 2007; Ghosh, 2009a), although in some cases virucidal activity plays a role

(Ghosh, 2009a). However, the native sulfated xylomannan of S. polydactyla did not exert

any inactivating effect against HSV-1 allowing to discard a direct virucidal action.

2-4. CONCLUSIONS

The present study highlight several novel and important aspects of S. polydactyla

derived polysaccharides with regard to their anti HSV properties and structural features

(i) A water soluble xylomannan containing 0.6 sulfate group per monosaccharide residues

and a molecular mass of 150 kDa could be easily generated from S. polydactyla with high

yield, (ii) This polymer contained a backbone of -(1 3)-linked D-mannopyranosyl

residues substituted at position 6 with single stub of -D-xylopyranosyl residues, (iii) the

chemically modified macromolecules which exerted high biological activity could be


35 | P a g e

analyzed in cell culture-based assay systems at a low level of cytotoxicity, (iv) strong

inhibitory effect was demonstrated against HSV (IC50 of F1S4 was 0.35 ± 0.03µg/ml), (v)

the DS appeared to be an important hallmark of anti HSV activity and (vi) the main mode

of action was directed to viral entry.

Chapter 3

ANTI CYTOMEGALOVIRUS ACTIVITY AND STRUCTURAL FEATURES OF SULFATED GLUCANS PREPARED FROM A COMMERCIAL SOURCE OF RICE BRAN (Oryza sativa)

Anti-cytomegalovirus activity of sulfated glucans: Chapter 3

37 | P a g e

3-1. INTRODUCTION

Human cytomegalovirus (HCMV), the type species of cytomegaloviruses in the

β-subfamily of Herpesviridae, has the regulatory capacity to initiate on the one hand a

productive replication upon primary infection and on the other hand to maintain a

lifelong persistence even within an entirely immunocompetent host (Mocarski, 2007).

From a latent state, HCMV may periodically reactivate without causing symptoms in

healthy individuals. In the absence of an adequate host immune response, the fine-

balanced host-virus interaction is shifted to an extreme situation in which HCMV can

eventually cause invasive disease and indirect immunopathogenesis-associated effects

(Crough and Khanna, 2009). This opportunistic pathogen is associated with significant

morbidity and mortality among immunocompromised patients (in particular

immunosuppressed patients after stem cell or solid organ transplantation as well as

immunocompromised acquired immune deficiency syndrome or cancer patients).

Additionally, congenital HCMV infection, occurring in 1 - 2% of all live births, is a

leading cause of birth defects and pathogenic courses of infection in infants. Drugs

currently available for the treatment of HCMV-associated disease comprise ganciclovir,

its oral prodrug valganciclovir, cidofovir, foscarnet and fomivirsen. Although these drugs

proved to be successful in the management of specific situations of HCMV disease, their

use is often limited due to toxic side-effects, poor oral bioavailability (with the exception

of valganciclovir) and the development of drug resistance. Furthermore, no drug has been

licensed for use in the treatment of congenital HCMV so far. Therefore, there is a need to

develop new compounds with a strong anti cytomegaloviral activity accompanied by

favourable pharmacological and clinical properties. The search for novel inhibitors of the


38 | P a g e

replication of cytomegalovirus and other herpesviruses has led to the identification of

additional molecular targets such as viral glycoproteins (Adamiak, 2007; Cheshenko and

Herold, 2002; Shukla, 1999) and putative cellular entry receptors such as platelet derived

growth factor receptor (reviewed by Marschall and Stamminger, 2009). Herpesviral

glycoproteins may interact with both, entry receptor proteins and specific sites on

heparan sulfate (HS) (Geraghty, 1998; Montgomery, 1996; O’Donnell, 2006; Shukla,

1999; Xu, 2005) .The uniquely distributed sulfation pattern of HS is believed to regulate

its functional specificity (Gama, 2006; Liu, 2007). Interestingly, heparin and several

sulfated carbohydrates including sulfated fucans, galactans, xylans and xylomannans

have been found to inhibit herpesviral entry, e.g. entry of herpes simplex virus, because

of their structural similarity to HS (Balzarini and Van Damme, 2007; Ghosh, 2009a;

Witvrouw and De Clercq, 1997). However, the naturally occurring sulfated

polysaccharides are mostly complex and heterogeneous which may complicate their use

in medical applications. Hence, these complex macromolecules have the potential to bind

to a variety of physiologically important proteins and thereby harbour an intrinsic risk to

induce off-target effects and cytotoxicity. This risk, however, may be reduced by using

structurally defined polysaccharides binding to their ligands with higher specificity. With

this regard, procedures of experimental sulfation are an important improvement in the

generation of structurally defined polysaccharides exerting high biological activity.

Currently, huge amounts of rice bran (Oryza sativa), a by-product of the rice-

processing industry, is regularly disposed as waste which might be utilized profitably as a

source of various pharmaceutical products. Rice bran biomass retains heavy metals

(Montanher, 2005), has anti oxidation activity (Jang and Xu, 2009) and may be used for


39 | P a g e

the removal of selected organics from water (Akhtar, 2005). The diversity of

polysaccharides massively contained in rice bran is the basis for a number of very

interesting biological activities (Cholujova, 2009; Ghoneum, 2005). However, the full

exploitation of these polysaccharides requires an in-depth knowledge of their structure-

activity relationships.

The present study reports the generation and chemical characterization of glucans

from rice bran. Using chemical and chromatographic methods as well as various forms of

spectroscopy, we have been able to deduce structural features of this polysaccharide. The

possibility to generate derivatives by chemical sulfation in the O-positions along the

polysaccharide has led to the identification of a series of highly interesting, related

compounds possessing varied degrees of sulfation and charge distribution. An analysis of

these compounds in cell culture-based assay systems indicated a substantial anti viral

activity in a non-cytotoxic range of concentrations. In particular, a strong anti

cytomegaloviral effect could be demonstrated. The putative activity-defining components

of this group of antiviral substances are discussed on the basis of our structure-activity

determination.

3-2. CHEMICAL CHARACTERIZATION OF GLUCANS

3-2.1. Preparation of de oiled rice bran and sugar composition analysis

The major goal of this study was to develop antiviral drug candidate from rice

bran. Commercial rice bran preparation contains small amounts of oil which interfere

during extraction of polysaccharides. Therefore, the commercial bran was sequentially


40 | P a g e

Fractions Rb RbWE RbG S1G S2G S3G S4G

Total Sugar, %a 61 69 64 29 31 32 33

Uronic Acid, %a 0.6-0.8 Tr 0.3-0.4 Tr Tr Tr Tr

Degree of Sulfationb Nd Nd Nd 2.0 1.9 1.8 1.7

Arabinose, %c 1 2 Tr Tr Tr Tr Tr

Xylose, %c 5 6 8 9 7 8 9

Mannose, %c 1 Tr Tr Tr Tr 4 9

Galactose, %c Tr Nd Nd Nd Nd Nd Nd

Glucose, %c 94 92 92 91 93 92 92

Fig. 3.1. Scheme for the preparation of de oiled rice bran available from commercial source.

De oiled rice bran

Removal of oil on treatment with petroleum ether and

acetone

Rice bran Oryza sativa

Table 3.1. Sugar composition of the de oiled rice bran and the fractions generated therefrom

The sugar composition (molecular %) was determined for the following fractions: de oiled

rice bran (Rb), the water-extracted polymer (RbWE), rice bran glucan (RbG) and sulfated

derivatives of glucan obtained at different time points in the sulfation process (S1G, S2G, S3G

and S4G at 4.0, 3.5, 2.5 and 2.0 h, respectively). aPercent weight of fraction dry weight (w/w).

bNumber of sulfate group per monosaccharide residue. cMolecular percentage of neutral

sugars. Nd, not determined. Tr, trace.


41 | P a g e

extracted in a Soxhlet apparatus with petroleum ether and acetone to leave de oiled Rice

bran (Fig 3.1). In the next step, sugar composition of the de oiled rice bran was

determined. This information is important as it helps to understand the type of

polysaccharides present.

The bran used contained 61% (w/w) sulfuric acid (2M) hydrolysable

polysaccharides of which a minor component (0.6 – 0.8%) was uronic acid. The main

neutral saccharide was glucose (Table 3.1), but xylose and arabinose were also released.

3-2.2. Isolation and degradation

This de oiled rice bran was extracted with water (Fig 3.2) to get water extracted

polysaccharide containing fraction RbWE. Purification of the water-extracted fraction

was then achieved by repeated precipitation of the macromolecule from solution with

dehydrated ethanol (water extract : ethanol :: 1 : 4).

The hot water extracted materials (RbWE) amounted to 12% of the starting bran

preparation and contained 69% polysaccharides on the basis of fraction dry weight

(Table 3.1). It was composed mainly of glucose. Xylose and arabinose were the two

other monosaccharides found in small quantities. No amino sugars were detected during

GLC-MS analysis of the derived alditol acetates. Thin layer chromatographic (TLC)

analysis of the monosaccharides present in the hydrolysate indicates the presence of, inter

alia, an uronic acid with Rf value similar to that of glucuronic acid.

A so far poorly exploited potential of polysaccharides is their frequently reported

ability to interfere with both, very early and very late steps in viral replication, such as


42 | P a g e

entry and release (reviewed by Ghosh, 2009a). As far as the inhibition of viral release and

spread is concerned, small-sized oligosaccharides were shown to affect cell-to-cell spread

of viruses more efficiently than their high-molecular mass counterparts (Nyberg, 2004).

Therefore, we have decided to generate polysaccharides with comparably low molecular

mass from the water extracted rice bran material (RbWE). Based on the results of pilot

experiments, we have hydrolyzed the RbWE fraction with 100 mM of HCl to yield a

fraction termed RbG. The total polysaccharide content of RbG fraction was 64% on the

basis of fraction dry weight (w/w) and the major sugar was glucose (Table 3.1).

Fig. 3.2. Scheme for the isolation and sulfation of glucans from a commercial rice bran

preparation.

De oiled rice bran (Rb)

Water Extract

(RbWE)

Extraction with water, pH 6.5 - 7.0, 90°C, 1h x 3

S1G S2G S3G S4G

Sulfation using SO3- pyridine

complex, 30°C

(DEAE-Sepharose FF)

4.0 h 3.5 h 2.5 h 2.0 h

Hydrolysed with 100 mM HCl, 90°C, 2 h

Rice bran Glucan

(RbG)

Reaction time:


43 | P a g e

3-2.3. Generation and purification of sulfated glucans

As a general rule, the degree of sulfation (DS) has major impact on the antiviral

activity of polysaccharides (Witvrouw and De Clercq, 1997). Therefore, a high charge

density appears to be favourable for high antiviral activity, at least for particular classes

of polysaccharides (Ghosh, 2009a).

In addition to this well-documented

impact of DS, the specific position

of the sulfate group also reported to

be important for antiviral activity of

the various sulfated glycans (Ghosh,

2009a; Copeland, 2008). To

investigate the specific effect of DS,

we have sulfated RbG under various conditions to yield sulfated derivatives S1G, S2G,

S3G and S4G. These sulfated glucans were purified by size exclusion chromatography on

SuperdexTM

30 prep grade column. Fractions eluted at Kav. value 0.3–0.6 were pooled and

lyophilized. The polysaccharide content and their specific DS are given in Table 3.1.

Solutions of sulfated glucans (S1G, S2G, S3G and S4G) in 500 mM sodium

acetate buffer (pH 4.0), were loaded to a SuperdexTM

30 prep grade column equilibrated

with the same buffer (Fig 3.3). It consisted mostly of glucose together with trace amount

of xylose and arabinose (Table 3.1). The sugar composition of S1G was similar to S2G,

although its yield (15%) was higher. The difference in the elution between S1G and S2G

was probably due to the level of uronic acid and sulfate. The column was eluted

ascendingly with the same buffer at 15 ml/h, and the temperature was 30 – 35°C. The

Yie

ld %

S1G

S2G

S3G

S4G

Fig. 3.3. Yield percentages of different fractions purified by SEC on SuperdexTM 30 prep grade column.


44 | P a g e

column was calibrated with standard dextrans within a molecular-weight range of 1,000

to 30,000 Da.

All sulfated derivatives (S1G, S2G, S3G and S4G) were collected from

SuperdexTM

column and injected separately on a SephadexTM

G-10 column (2.6 x 90 cm;

Amersham Pharmacia biotech AB, Uppsala, Sweden). The desalted materials were

concentrated and then lyophilized.

3-2.4. Molecular mass

Usually antiviral activity of sulfated polysaccharides correlates with the

molecular mass of the chain. The higher the average molecular weight, the higher is the

anti viral activity in many cases. So we wanted to determine the molecular mass of the

sulfated glucan (S1G) by Size exclusion chromatography (SEC). All sulfated fractions

were submitted to SEC onto Superdex 30TM

prep grade column. As indicated by the Kav.

values, all fractions eluted within the fractionation range of the column. These

polysaccharides were polydisperse, as expected for acid generated fragments. Fractions

eluted at Kav. value 0.3 – 0.6 were pooled and lyophilized. Based on calibration with

dextrans, the apparent molecular weight of the S1G peak would be 5,000 Da. It should be

noted, however, that molecules containing sulfate groups may have a different

hydrodynamic volume than dextrans due to intramolecular electrostatic repulsions caused

by charge effects and may therefore elute at a different rate than expected on the basis of

their molecular weight.


45 | P a g e

3-2.5. IR-Spectroscopy

Fig. 3.4. Fourier transform-infra red spectra of RbG and its sulfated derivatives

A

4000 3500 3000 2500 2000 1500 1000 500

0

1

2

3

4T

ran

smit

tan

ce,

%

Wave number, cm-1

4000 3500 3000 2500 2000 1500 1000 500

4

5

6

7

8

9

10

11

12

13

14

15

16

17

Tra

nsm

itta

nce

, %

Wave number, cm-1

S4G

S1G

818 828

1260

B

(A) Rice bran glucan (RbG) generated from a commercial rice bran preparation. (B) RbG

sulfated derivatives (S1G and S4G). Absorption bands at 1260cm-1 are assigned to the

stretching of the S=O bond, whereas bands at approximately 818 cm-1 and 828 cm-1

were ascribed to the C-O-S stretching 6-O-sulfate groups, and equatorial 2-O- & 3-O-

sulfate groups of glucose residues, respectively.


46 | P a g e

Sulfate group plays an important role in anti viral activities and its presence can

be determined with the help of Fourier Transform (FT)-IR spectroscopy (Adhikari, 2006;

Bandyopadhyay, 2011; Franz., 2000; Gunay and Linhardt, 1999; Ray, 2011; Saha, 2011;

2012; Sinha, 2010). IR spectra of the rice bran glucan RbG and its O-sulfated derivatives

S1G and S4G (Fig 3.4) strongly suggest the conversion of hydroxyl groups to O-sulfate

groups. The intensity of the absorbances at 1260 cm-1

and 800 – 850 cm-1

attributed to the

stretching of S=O bond and C–O–S bonds, respectively, are dramatically increased by O-

sulfation. Similarly, the intensity of the bands at 2932, 1424, 1370 and 1004 cm-1

,

attributed to the stretching and or deformation vibration of C–O–H bonds, are decreased

in the spectrum of the O-sulfated glucan sulfate. Assignments of IR absorption bands at

1260 cm-1

and in the 800 – 850 cm-1

spectral region were based on the principle originally

published by Orr (1954) and Lloyd (1961). Bands at about 818 cm-1

were tentatively

ascribed to 6-O-sulfate groups based on the report of Grant (1991), and the band at 828

cm-1

was ascribed to C–O–S stretching of equatorial 2-O and 3-O-sulfate groups of

glucose residues based on reports by Lloyd (1961), and Lloyd and Dodgson (1961).

3-2.6. Linkage analysis

Anti viral activity of these macromolecules depends on their structures (Ghosh,

2009). So we wanted to determine the glycosidic linkages of the constituent sugars by

methylation analysis. Glycosyl linkage analysis of the glucan (RbG) indicated the

presence 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylglucitol residue suggesting that the polymer

is linear and possesses a 1,4-linked backbone (Table 3.2).


47 | P a g e

Analysis of S4G showed five different partially methylated alditol acetates

indicating the presence of sulfate groups at various positions. The appearance of 1,4,6-

linked residues suggests the presence of 1,4-linked glucopyranosyl units that are O-

sulfated only at C6 (Fig 3.5). Similarly, the presence of 1,2,4,6- and 1,3,4,6-linked

glucopyranosyl residues suggests the presence of 2,6- and 3,6-disulfated residues,

respectively. Another striking feature of this analysis is the presence of 1,4-linked

residues. This may indicate that some of the glucopyranosyl residues are non-sulfated. In

the present study, methylation analysis was carried out under strongly basic condition

which may have induced partial desulfation (particularly at C6 positions), possibly

followed by a conversion of the hydroxyl groups into their methyl ethers.

Methylation

products

m/z values

Peak area*

RbG S1G S4G

2,3,6-Glcp† 43, 102, 115, 118, 131, 162, 175 and 233 100 Nd 16

2,3-Glcp‡ 43, 118, 201 and 261 Tr Nd 27

3-Glcp* 43, 115, 130, 190, 201, 261and 333 Nd Nd 26

2-Glcp# 43, 118, 187, 202 and 333 Nd Nd 18

Glcp± 43, 115, 139, 170, 187, 217, 259, 289 and

361

Tr 100 13

*Percentage of total area of the identified peaks. †1,4,5-tri-O-acetyl-2,3,6-tri-O-methylglucitol. ‡1,4,5,6-tetra-O-acetyl-2,3-di-O-methylglucitol.*1,2,4,5,6-penta-O-acetyl-3-O-methylglucitol. #1,3,4,5,6-penta-O-acetyl-2-O-methylglucitol. ±1,2,3,4,5,6-hexa-O-acetylglucitol. Nd = not

detected. Tr = trace.

Table 3.2. Partially methylated alditol acetates derived from Rice bran glucan (RbG) and its

sulfated derivative (S1G and S4G)


48 | P a g e

C

x 1

2

3

4

6

5

5

B

x

1

6

A

Retention time, min

6

50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.00.0

5.0

10.0

15.0

20.0

25.0

%43 118

10285

4259 111 261201142 162 187177 233 305219

50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 350.00.0

5.0

10.0

15.0

%43

130

9985

190

60 261115 201159141 217171 245 275 333

Fig. 3.5. GLC –MS analysis of partially methylated alditol acetates (PMAA) derived from (A) Rice

bran glucan (RbG) and its sulfated derivatives (B) S1G and (C) S4G. These

macromolecules were completely methylated and then hydrolyzed, reduced and

converted into their partially methylated alditol acetates. 11,4,5-tri-O-acetyl-2,3,6-tri-O-

methylglucitol. 21,4,5,6-tetra-O-acetyl-2,3-di-O-methylglucitol. 31,3,4,5,6-penta-O-acetyl-

2-O-methylglucitol. 41,2,4,5,6-penta-O-acetyl-3-O-methylglucitol.51,2,3,4,5,6-hexa-O-

acetylglucitol. 6internal standard (myo-inositol). ximpurity.


49 | P a g e

Appearance of 1,2,3,4,6-linked glucopyranosyl residues suggests the presence of

2,3,6-trisulfated residue, but complete methylation of sulfated polysaccharides is

generally difficult as the steric hindrance and electronic effect exhibited by the sulfate

esters represents a limitation (Adhikari, 2006; Patankar, 1993; Pereira, 1999). Finally,

analysis of S1G revealed exclusively a single peak corresponding to glucitol hexa-

acetate, which indicates that this compound may have been completely sulfated (Table

3.2).

Notably, the partially methylated alditol acetates (PMAA) were separated by

GLC and identified by their (i) retention time relative to that of inositol hexa-acetate

(internal standard) and (ii) mass spectral fragmentation patterns. Mass spectral (EI)

fragmentation pattern of 1,2,3,4,5,6-hexa-O-acetylglucitol, 1,3,4,5,6-penta-O-

acetyl-2-O-methylglucitol, 1,2,4,5,6-penta-O-acetyl-3-O-methylglucitol, 1,4,5-

tri-O-acetyl-2,3,6-tri-O-methylglucitol and 1,4,5,6-tetra-O-acetyl-2,3-di-O-methylglucitol

are given below (Fig 3.6).


50 | P a g e

C H O A c

C H D O A c

C H OAc

C H2OAc

C H O Me

C H OAc

190 130

261

201

50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.0 350.00.0

5.0

10.0

15.0

%43

130

9985

190

60 261115 201159141 217171 245 275 333

(4) 233 118 162

173

102

C H O A c

C H D O A c

C H O M e

C H2OMe

C H O M e

C H O A c

m/z 50 75 100 125 150 175 200 2250e3

100e3

200e3 43

118

10287233

13175 16259 173142 203191 235

50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.0 325.00

10

20

30

40

50

%43

118

45 97 1398774 160 333259202145 187 217 231 245 273 299

(2)

m/z

C H O A c

C H D O A c

C H O M e

C H2OAc

C H OAc

C H OAc

118

(3)

m/z

50 100 150 200 250 300 3500

25000

50000

43

115

139

103 187 217157170 25973 28941 361

(1)

m/z


51 | P a g e

3-2.7. NMR spectroscopy

NMR spectroscopy is a convenient method to give valuable structural information

of polysaccharides (Bandyopadhyay, 2011; Ray, 2011; Pereira, 1999; Saha, 2012). We

employed NMR analysis to determine the anomeric configuration and the sulfation

pattern of the RbG sulfated derivatives.

The 1H-NMR spectrum of the glucan fraction (RbG) shows the anomeric signal

(H1) at 5.42 ppm and ring protons (H2 to H6) at 3.6 – 4.0 ppm characteristic of –glucan

(Fig 3.7 A). The 13

C-NMR spectrum of a sulfated glucan fraction (S4G) was measured

(Fig 3.7 B) and signals were assigned by comparison with data published for maltose,

amylose, amylopectin and a sulfated glucan from alga (Bock and Thogersen, 1982;

Lechat, 2000). The C6 signal of α-(14)-linked glucopyranosyl residue appears around

60 – 61 ppm (Dais and Perlin, 1982). Besides this, it has been reported that O-sulfation of

Fig. 3.6. EI-MS fragmentation pattern of partially methylated alditol acetates derived from

Rice bran glucan (RbG). (1) 1,2,3,4,5,6-hexa-O-acetylglucitol; (2) 1,3,4,5,6-penta-O-

acetyl-2-O-methylglucitol; (3) 1,2,4,5,6-penta-O-acetyl-3-O-methylglucitol; (4) 1,4,5-

tri-O-acetyl-2,3,6-tri-O-methylglucitol; (5) 1,4,5,6-tetra-O-acetyl-2,3-di-O-

methylglucitol.

(5)

m/z

C HOAc

C H D O A c

C H O Me

C H2OAc

C H OMe

C H OAc

118 261

201

50 75 100 125 150 175 200 225 2500e3

10e3

20e3 43

118

99

8512759 71 261187150 167137 201


52 | P a g e

α,β-D-glucose causes a strong, downfield shift (+6.5 to +9.5 ppm) of the substituted

resonances that is accompanied by smaller, up- or down-field displacement (-2.2 to +0.2

ppm) of the signals of adjacent carbon atoms (reviewed by Gorin, 1981). The presence of

signals around 67 – 68 ppm and absence of signals at 61 – 62 ppm suggests that all

Fig. 3.7. Spectra of RbG and S1G

HOD

α-H1

H2 - H6

Chemical shifts, ppm

Chemical shifts, ppm

C6

S

C2

C 1

C2

S

C3

C

5

C4

C3

S

A

B

(A) 1H-NMR spectrum of the rice bran glucan (RbG). Integrals for each signal are shown in

vertical columns. (B) 13C-NMR spectrum of the RbG sulfated derivative (S1G). The

spectra were recorded at 25°C in D2O solution. The chemical shifts are reported

relative to an internal acetone standard at 2.225 and 31.55 ppm for 1H- and 13C-NMR

spectra, respectively. HOD, the signal for deuterated water


53 | P a g e

hydroxyl groups at the position C6 of the glucans analyzed in the present study were

sulfated. This was considered as the expected situation since the hydroxyl group at C6

position is primary whereas those at C2 and C3 are secondary. The chemical shift of C6

is in good agreement with that of algae-derived sulfated glucans (67.6 ppm). In maltose,

amylose, amylopectin and algal glucans, the signal of C2 is usually downfield from the

C3 signal, which in its turn downfield from C5 (Lechat, 2000). Therefore, signals around

76, 74 and 73 ppm in our analysis may tentatively be attributed to the C2, C3 and C5,

respectively, of mono-sulfated glucose residues that are O-sulfated at C6. Another

striking characteristic in the chemical shifts of values are the downfield shifts in C2 and

C3 that may indicate their participation in O-sulfation. Taken together, signals at 82.8 and

81.4 ppm could tentatively be assigned to the C2 and C3 of the 2,6- and 3,6-disulfated

residues. Comparison of the intensities of these signals with C2 and C3 signals of mono-

O-sulfated residues suggests that almost half of the glucose residues were not O-sulfated

at C2 and C3 positions. The chemical shift values (79 – 80 ppm) of the signals of C4

indicate its participation in the glycosidic linkage (see similar shift to 79.9 ppm) in the

sulfated glucans derived from algae (Lechat, 2000).

3-3. BIO-ASSAY OF RICE BRAN DERIVED SULFATED

GLUCANS

3-3.1. Anti cytomegalovirus activity

The preparations of sulfated polysaccharides were analyzed for antiviral activity.

For this purpose, an established cell culture-based assay for quantifying the replication of

HCMV was applied which proved to be highly reliable for the screening of novel


54 | P a g e

Fig. 3.8. Inhibition of HCMV replication by sulfated glucans.

Data are shown as mean ±SD. Primary human foreskin fibroblasts (HFFs) were used for

infection with human cytomegalovirus (HCMV) strain AD169-GFP (0.25 GFP-FU/ml). Antiviral

substances were added at various time points of infection: during preincubation of the cells (1

h) with substances before addition of virus (pre); with substances present during virus

adsorption (90 min; ads); with substances continuously incubated postadsorption (post); and

with substances constantly present (pre-ads-post). After 7 days, cells were lysed and used for a

quantitative determination of virus replication by automated GFP fluorometry. (A) Various

concentrations of sulfated rice bran glucan S1G were analyzed for anti HCMV activity under

pre-ads-post conditions. (B) Sulfated rice brain glucans S1G and S2G (270 μg/ml) were

analyzed under various conditions as indicated. (C) Inoculum virus was pretreated with S1G

for 1 h at room temperature before virus particles were pelletized by centrifugation and used

for infection of HFFs in the absence of S1G (as an inhibitor control, GCV was incubated on cells

under post conditions). All values represent the mean of determinations in quadruplicate.

Mock-infection, no infection, No inhibitor, HCMV infection in the absence of inhibitor.


55 | P a g e

antiviral substances (Marschall, 2000; Sampaio, 2005; Herget, 2004; Rechter, 2006;

Schleiss, 2008). Amongst all substances analyzed, S1G and S2G exerted a particularly

strong and concentration-dependent inhibitory activity towards HCMV replication (Fig

3.8 A-B). No sign of cytotoxicity was observed for these substances in the analyzed range

of concentrations (determined by microscopic inspection of cells and testing of

cytotoxicity using the CytoTox 96 Non-Radioactive Cytotoxicity Assay, Promega; data

not shown). The lowest 50% Inhibitory Concentration (IC50) of anti HCMV activity was

determined for S1G with 3.46 ± 0.63 µg/ml. Importantly, these hypersulfated glucans

(S1G and S2G) showed clear anti cytomegaloviral potential, while the non-sulfated

parental substance (RbG) was negative (Fig 3.9 A). Thus, these data support the

previously stated notion that the DS is one of the determinants conferring high antiviral

activity to polysaccharides.

3-3.2. Viral entry is the main mode of activity of sulfated glucans S1G and

S2G

As a characteristic feature of the anti cytomegaloviral activity of rice bran-derived

sulfated glucans S1G and S2G, virus inhibition was primarily achieved when the

substances were preincubated on the cells (added 1 h prior to HCMV infection) and/or

were present during the virus adsorption phase. When these substances were not present

during this initial period of the experiment, but added at later time points, e.g. directly

after the virus adsorption phase, a very poor or no inhibitory effect was measured (Fig

3.8 B, central bars). In particular, the preincubation of S1G and S2G alone was sufficient


56 | P a g e

to produce a substantial antiviral effect (Fig 3.8 B, left-side bars). The most pronounced

antiviral activity of these polysaccharide substances, however, was measured when they

Fig. 3.9. Comparative analysis of the replication of herpesviruses: potential inhibitory effects of

experimentally hypersulfated glucan versus poorly sulfated glucan.

Cells were infected with (A) human cytomegalovirus (HCMV) strain AD169-GFP, (B) HCMV

strainTB40 UL32-EGFP, (C) varicella zoster virus (VZV) starin Oka or (D) Epstein-Barr virus (EBV)

strain B-95-8. Antiviral substances were pre-incubated on the cells (1 h) and remained present

during virus adsorption before replacement by fresh culture media. After 6-7 days, cells were lysed

and used for a quantitative determination of virus replication by automated green fluorescent

protein fluorometry (HCMV, EBV) or plaque staining (VZV). A mean of four values is presented in

each column (infection in duplicate; processing of lysates and measurement or microscopic plaque

counting in duplicate). GCV, ganciclovir (20 µM); mock-infected, no infection, No inhibition, HCMV

infection control in the absence of inhibitor; RbG, poorly sulfated glucan; S1G, hypersulfated glucan.


57 | P a g e

were used for preincubation and remained continuously present during virus replication

until the termination of the experiment (Fig 3.8 B, right-side bars). This feature pointed

to an inhibition of HCMV entry and penetration and stood in contrast to the well-

characterized inhibitory effect produced by the reference drug, ganciclovir (GCV), an

antagonist of viral genome replication. While GCV showed no inhibitory potential when

exclusively preincubated on cells and removed before the addition of virus inoculum,

later time points of addition were highly effective. This finding demonstrated a marked

mechanistic difference between GCV and the sulfated glucans S1G and S2G. To address

the question whether the measured antiviral activity might have resulted from a direct

virucidal effect, S1G was preincubated with inoculum virus before infection. After 1 h,

infectious virus particles were separated from the substance by high-speed centrifugation

(3 h at 14.000 rpm in Eppendorf centrifuge) and subsequently used for the infection of

cells. Under these conditions, no significant inhibitory effect could be determined up to

the highest concentration analyzed (270 µg/ml), indicating that virucidal activity did not

play a major role in the anti cytomegaloviral potency of S1G (Fig 3.8 C). Moreover, an

experiment was performed to analyze a possible inhibitory activity of S1G on viral cell-

to-cell spread. For this purpose, virus infection (HCMV AD169-GFP) was performed in

the presence of a neutralizing human immunoglobulin preparation (Biotest HIg 6030398;

kindly provided by Michael Mach and Barbara Kropff, Erlangen) or a non-neutralizing

control serum (rabbit) which were both added to the culture media after viral entry. Seven

days post-infection, viral replication was quantified by measuring the GFP signals

expressed (by GFP microscopy and fluorometry). When S1G had been added to the cells

after viral entry (post conditions), inhibition of HCMV was very inefficient, both under


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suppressed levels of viral spread by neutralizing HIg as well as in the control without

neutralizing HIg. Hereby, no reduction in viral plaque size was noted, indicating that

effects on cell-to-cell spread were poor or missing (data not shown). When S1G was

incubated throughout the infection (pre-ads-post conditions), inhibition was very efficient

in both cases, thus confirming the earlier statement that the main mode of anti

cytomegaloviral activity was directed to viral entry.

3-3.3. Antiviral activity of S1G is selectively detectable for human

cytomegalovirus but not for other human herpesviruses

The question was addressed whether S1G and possibly related rice bran-derived

sulfated polysaccharides exert an antiviral activity selective or non-selective for HCMV,

i.e. whether S1G produces a similar inhibitory effect towards other human herpesviruses.

To this end, a comparative analysis was performed using HCMV, VZV and EBV. As a

striking result, S1G selectively inhibited HCMV but was entirely ineffective against VZV

and EBV (Fig 3.9 A-D). The anti HCMV activity was independent from viral strain since

very similar inhibitory effects were determined for strains AD169 and TB40 (Fig 3.9 A-

B; GFP-expressing recombinants of the two viral strains were used to quantify viral

replication). Notably, in the range between 1.1 µg/ml and 90 µg/ml, a very pronounced

selectivity of this sulfated glucan for HCMV could be demonstrated. In comparison, the

non-hypersulfated parental preparation RbG was negative for all four viruses analyzed,

thereby stressing the significance of DS of these substances for antiviral activity. The

mode of anti HCMV activity of S1G and the molecular basis of selectivity will have to be


59 | P a g e

determined to learn more about this highly interesting group of rice bran-derived

substances.

3-4. CONCLUSIONS

In summary, the findings of this study highlight several novel and important

aspects of the commercial rice bran preparation-derived polysaccharides with regard to

their antiviral properties. (i) A number of water soluble polysaccharides could be easily

generated from commercially available rice bran preparation, (ii) these substances

exerted high biological activity which could be analyzed in cell culture-based assay

systems at a low level of cytotoxicity (undetectable up to the concentration of 270

µg/ml), (iii) strong antiviral effect was demonstrated for HCMV (IC50 of S1G 3.46 ± 0.63

µg/ml), (iv) the DS appeared to be an important hallmark of anti HCMV activity, (v) the

main mode of action was directed to HCMV entry, and (vi) amongst three human herpes

viruses investigated, the antiviral activity of S1G was selective for HCMV. Further

studies will be needed to characterize the mode of antiviral activity in detail and to define

the chances of sulfated polysaccharides for their putative usefulness for topical

administration in antiviral therapy (for review see Ghosh, 2009a).

Experimentals

Chapter 4

MATERIALS

AND

METHODS

Materials and Methods: Chapter 4

62 | P a g e

4-1. PLANT MATERIALS AND PRELIMINARY TREATMENTS

4-1.1. Red seaweed Sebdenia polydactyla

Samples of Sebdenia polydactyla were collected from the Okha coast of Gujarat,

India in August 1995. The seaweeds were washed thoroughly with tap water, dried by forced

air circulation and pulverized in a Waring Blender. After pulverization in a commercial

grinder, Depigmentation of 132 g of algal powder was done using sequential extraction with

petroleum ether and acetone as solvent in a Soxhlet apparatus. The unextracted material was

placed in a plastic beaker, and air dried to yield depigmented algal powder (DAP, 91 g).

4-1.2. Commercial preparation of rice bran (Oryza sativa)

The commercial rice bran preparation (Batch No. G5458) was a gift from Laxmishree

Rice Mill, Burdwan,West Bengal, India.

4.2. EXTRACTION OF POLYSACCHARIDES

4-2.1. Xylomannans from the red seaweed Sebdenia polydactyla

DAP (10 g) was extracted thrice with water (1:100) (pH 6.0) at 25-32°C, under

constant stirring (12 h, three times). Separation of the residue from the liquid extract was

performed by centrifugation followed by filtration through glass filter (G-2). The residue was

briefly washed with additional distilled water and the wash was collected to maximize

polysaccharide recovery. The liquid extract was dialyzed extensively against water and

lyophilized. The recovered material was dissolved in water; the polysaccharides were


63 | P a g e

precipitated twice with ethanol (4 vol.) and then collected by centrifugation. The final pellet

was dissolved in water and lyophilized to yield the water extracted polysaccharide,

designated as WE (3.4 g). The aqueous 80% ethanol soluble fractions were combined,

desalted on SephadexTM

G-10 column (2.6 x 90 cm; Amersham Pharmacia biotech AB,

Uppsala, Sweden) and finally lyophilized to produce WE-S fraction.

4-2.2. Glucans from a commercial preparation of rice bran (Oryza sativa)

The commercial bran (Rb, 10 g) was extracted three times with water (pH 6.5–7.0)

at a solute to solvent ratio of 1:100 (w/v) at 90°C for 1 h under constant stirring. Separation

of the residue from the liquid extract was performed by centrifugation (Research Centrifuge

R-24, Remi, Mumbai, India) followed by filtration through a G-2 glass filter (Borosil Glass

Works Ltd., Kolkata, India). The residue was briefly washed with additional distilled water

and the wash was collected to maximize polysaccharide recovery. The liquid extract was

dialyzed extensively against water and lyophilized (Cool Safe 55-F Freeze drier, Scanvac,

Denmark). The recovered material was dissolved in water; the polysaccharides were

precipitated twice with ethanol (4 vol.) and then collected by centrifugation. The final pellet

was dissolved in water and lyophilized to yield the water-extracted polysaccharide, named

RbWE (1.2 g).

The glucan-rich pool (RbWE) was treated (0.5 mg/ml at 90°C for 2 h) with 100 mM

HCl. After incubation, the resulting digest was cooled, neutralised with 2 M NaOH, dialyzed

(Sigma-Aldrich, USA, Molecular cut-off 1,000 Da) and then lyophilized (RbG).


64 | P a g e

4-3. ANALYTICAL METHODS

4-3.1. General

All experiments were conducted at least in duplicate, the mean and standard deviation

was directly calculated from the functions present in excel program. Evaporations were

carried out under reduced pressure at around 45°C. Dialysis against distilled water was

performed with continuous stirring, toluene being added to inhibit microbial growth.

Moisture was determined by drying ground material in an air-circulated oven at 110°C for

3 h.

Chemicals used were analytical grade or best available. Except otherwise stated data

presented are the mean values of at least three independent analyses. Experiments were

repeated when the standard deviation was greater than 5%.

4-3.2. Sugar analysis

Total sugars were estimated as anhydroglucose by the phenol-sulphuric acid assay

(Dubois, 1956). Depending on sugar composition, the evaluation was corrected afterwards.

Total uronic acids were assayed as anhydrogalacturonic acid using m-phenyl phenol color

reagent (Ahmed and Labavitch, 1977).

Polysaccharides (10-12 mg) were hydrolyzed with 1 M sulfuric acid (3 h, 100°C) for

measurement of individual neutral sugar. Myo-Inositol was used as internal standard, and

hydrolytic losses were accounted for by using an external standard (Chatterjee, 2012).

Monosaccharides were reduced with sodium borohydride in anhydrous dimethyl sulfoxide.

After reduction, excess sodium borohydride was decomposed by the addition of acetic acid.

Acetylation was carried out by acetic anhydride and 1-methylimidazole was added as catalyst


65 | P a g e

(Blakeney, 1983). Derived alditol acetates were analyzed by GLC and GLC/MS (Sinha,

2011).

Alternatively, the samples were hydrolyzed using trifluoroacetic acid (2 M, 2 h at 110

°C), followed by an 18 h methanolysis at 80°C with dry 2 M methanolic-HCl. For water

insoluble residues TFA hydrolysis was followed by a treatment of the residue with 72%

(w/w) H2SO4 for 1 h at room temperature and then with 2 M H2SO4 for 2 h at 100°C.

Mannitol and / or myo-inositol were used as internal standard. The generated methyl

glycosides were converted into their TMS-derivatives using trimethylsilyl chloride and

separated by GLC (York, 1985).

4-3.3. Sulfation

Further sulfation of the purified xylomannan sulfate (F1) was carried out as described

(Chattopadhyay, 2008; Ghosh, 2010; Karmakar, 2010; Mandal, 2010; Saha, 2010; Sinha,

2010). Briefly, F1 (30 mg) and SO3-pyridine (45 mg) was dissolved in 0.5 ml dry DMF by

sonication and 50 l of dry pyridine was added to it. The mixture was heated in an oil bath at

90C in nitrogen atmosphere for 0.5, 1.0, 1.5 and 2.0 h. The solution was subsequently

neutralized with NaOH, dialyzed, and lyophilized to give the sulfated polysaccharides F1S1,

F1S2, F1S3 and F1S4, respectively.

Fraction RbG was sulfated using the method as described. Briefly, the freeze-dried

polysaccharide (100 mg) was soaked in 2 ml of dry N,N-dimethylformamide (DMF) and

SO3-pyridine complex dissolved in 12.5 ml DMF was mixed with the polysaccharide. For

every mol of SO3-pyridine complex, 1 mol of pyridine was added to the mixture. The

reaction was carried out under nitrogen atmosphere at 30C. The pH of the solution was then


66 | P a g e

adjusted to 7.0 with aqueous 20% NaOH. Finally, sulfated polysaccharides were dialyzed

against distilled water and freeze-dried. The sulfated derivatives S1G, S2G, S3G, and S4G

were obtained when the reaction times were 4.0, 3.5, 2.5 and 2.0 h, respectively.

4-3.4. Sulfate estimation by Turbidometric method

Samples (90 - 100 mg) were hydrolysed in 5 ml of 2 N hydrochloric acid in sealed

glass tubes at 100oC as described (Chattopadhyay, 2010). Sulfate was then determined using

a modified turbidometric barium chloride method (Craigie, 1984). The reagent was prepared

by dissolving 600 mg of gelatin in 200 mL of distilled water at 70oC and allowing the

solution to cool. After 16 h at 4oC the temperature was brought to 30 – 35

oC and 2.0 g of

BaCl2 .2H2O were added and dissolved. AR grade K2SO4 was used as a standard in the range

of 10 – 50 g S mL-1

. Accurately weighted samples (25 - 50 mg) were hydrolyzed for 2 h at

100oC in 0.75 mL of 2 N HCl in a sealed tube. The contents were then transferred and made

to volume in a 10 mL volumetric flask. Humic substances were removed by centrifugation. 2

mL of sample, 18 mL of distilled water and 2 mL of 0.5 N HCl was mixed in a 50 mL

Erlenmeyer flask. 1 mL of BaCl2-gelatin reagent was added and swirled. After 30 min

swirling again mixed the contents of the flasks and the turbidity was measured at 550 nm

against a reagent blank.

4-3.5. Desulfation

Desulfation was carried out by the procedure according to the procedure of Falshaw

and Furneaux (1998) as described by Karmakar, 2009. The pyridinium salt of the sample (1

g) was dissolved in DMSO containing 10% MeOH, and the solution was kept for 3 h at


67 | P a g e

100oC. The reaction mixture was cooled, diluted with an equal volume of water, and the pH

adjusted to 9.0 by the addition of 0.1 M sodium hydroxide. The solution was dialyzed against

running water for 20 h and lyophilization of the dialyzate gave desulfated sample.

4-3.6. Periodate oxidation and Smith degradation

Periodate oxidation of F1D of Sebdenia polydactyla was carried out according to the

method of Fry, 1988. Briefly, a solution of 100 mg of polysaccharide in 50 ml of reagent (50

mM NaIO4 made up in 0.25 M HCOOH, pH adjusted to 3.7 with 0.5 M NaOH) was

incubated in the dark for 144 h at 4 – 6°C. Next, the excess of periodate was decomposed

with 10 ml ethane-1,2-diol, and the solution stirred for a further 1 h period at room

temperature. To the vial containing oxopolysaccharide a solution (50 ml) of 950 mg of

NaBH4 in 1 M NaOH was added and the mixture was kept at room temperature for 12 h. The

solution was subsequently neutralized with CH3COOH (ice bath), dialyzed, and lyophilized

to give the oxidized and reduced polysaccharide. This preparation was hydrolyzed with TFA

(pH 2.0) for 10 min at 100oC and the resulting hydrolysate was desalted by passing through a

column (90 x 2.6 cm) of SephadexTM

G-10. Fractions (5 ml) were collected and analyzed for

their total sugar (Dubois, 1956) contents. Appropriate polymeric fractions were lyophilized

to afford a Smith degraded xylomannan, F1D-Sm (50 mg).

4-3.7. Linkage analysis

Methylation was carried out by the method of Blakeney and Stone (1985) as

described (Adhikari, 2006; Chattopadhyay, 2008; Ray and Lahaye, 1995). Exactly weighed

(1-2 mg) of the dried samples along with magnetic bars were taken in tubes fitted with air


68 | P a g e

tight rubber corks. Nitrogen atmosphere was maintained within the tubes. The samples were

dissolved in DMSO and to this solution were added lithium methyl carbanion (Stevenson and

Furneaux, 1991). The mixture was stirred in nitrogen atmosphere for 1 h. The tubes were

then cooled under ice bath and 0.4 ml ice cold methyl iodide was added, left the tubes for 1 h

at room temperature. The methylation process was repeated once more and the excess methyl

iodide was removed under vacuum. The resulting methylated polymer was then dialyzed and

lyophilized. The permethylated polysaccharide thus obtained were hydrolyzed with 2.5 M

trifloro acetic acid at 120°C for 75 min, reduced with 1 M NaBD4 in 2 M NH4OH for 3 h at

room temperature and acetylated with acetic anhydride using perchloric acid as a catalyst.

The partially methylated alditol acetates (PMAA) were analysed by GLC and GLC/MS as

described (Ray and Lahaye, 1995; Sinha, 2010). These compounds (PMAAs) were identified

by (i) measurement of relative retention times, (ii) methoxyl substitution pattern as obtained

from GLC-MS and (iii) carbohydrate composition of the non methylated polymers.

4-3.8. Chromatography

4-3.8.1. Thin layer chromatography (TLC)

The sugars released by acid hydrolysis (as described above in Section 4-3.2) were

also analyzed by TLC as described by Chatterjee, 2012. Briefly, TLC was carried out by

ascending technique using silica gel G impregnated with 0.5M NaH2PO4 as the supporting

material. Small amounts of samples were spotted for identification. The RF values of the test

material were compared with that of standard compounds to get an idea about the

components present and their purity. The color of the spot developed also conveys message

about its components.


69 | P a g e

The solvent system used for developing TLC was the following:

Ethanol: Phenol: Pyridine: 0.1 M Phosphoric acid :: 10 : 2 : 2 : 4

2-Propanol: Methanol: Water :: 16 : 1 : 3

Detection: By spraying saturated solution of aniline phthalate and heating the plate at 100oC.

4-3.8.2. Size exclusion chromatography (SEC)

System a: The water extracted fraction from S. polydactyla WE was chromatographed on a

Sephacryl S-300 column (2.6 x 90 cm; Amersham Biosciences AB, Uppsala, Sweden) using

0.5 M sodium acetate buffer (pH 5.0) as eluent. The flow rate of the column was 0.5 ml/min,

and fractions of 10 ml were collected and checked by the phenol–sulfuric acid reaction

(Dubois, 1956). The column was calibrated with standard dextrans (500, 70, 40 and 10 kDa).

System b: Solutions (3-5 ml) of sulfated glucans (S1G, S2G, S3G and S4G) in 500 mM

sodium acetate buffer (pH 4.0), were loaded to a SuperdexTM

30 prep grade column (2.6 x 60

cm; Amersham Pharmacia biotech AB, Uppsala, Sweden) equilibrated with the same buffer.

The column was eluted ascendingly with the same buffer at 15 ml/h, and the temperature was

30 – 35°C. The elution of glucans was expressed as a function of the partition coefficient Kav

[Kav = (Ve-V0)/(Vt-V0) with Vt and V0 being the total and void volume of the column

determined as the elution volume of glucose and blue dextran, respectively and Ve is the

elution volume of the sample]. The column was calibrated with standard dextrans within a

molecular-weight range of 1,000 to 30,000 Da. Fractions of 5 ml were collected and analyzed

for their total sugar contents by phenol-sulfuric acid method (Dubois, 1956) using glucose as

standard.


70 | P a g e

System c: All sulfated derivatives (S1G, S2G, S3G and S4G) were collected from

SuperdexTM

column and injected separately on a SephadexTM

G-10 column (2.6 x 90 cm;

Amersham Pharmacia biotech AB, Uppsala, Sweden). The desalted materials were

concentrated and then lyophilized.

4-3.8.3 Gas liquid chromatography (GLC)

GLC was carried out on

(i) GC-17A, Shimadzu and

(ii) HP6890 series Chromatograph.

The gas chromatograph was equipped with a flame ionization detector, the following

columns (i) – (iv) and helium as gas vector.

(i) WCOT fused silica capillary column (length 25 m, i.d. 0.25 mm) and film thickness

0.4m with CP-Sil 5 CP as stationary phase and helium as gas vector.

(ii) 3% SP-2340 on Supelcoport 100-120 mesh,

(iii) DB-225 (JW) and

(iv) SGE BP 225

The oven temperature program was:

(i) 210°C isothermal or

(ii) 2 min at 120°C, 10°C/min to 160°C, and 1.5°C/min to 220°C and then 20°C/min

to 280°C or

(iii) 170°C for 15min, 170-210°C at 5°C/min and 210°C for 15 min.


71 | P a g e

4-3.8.4. Gas liquid chromatography –Mass Spectrometry (GLC-MS)

The GLC-MS analysis were carried out using

(i) Shimadzu (Japan) GC 17 A coupled to QP 5050A MS and

(ii) GC (HP 6890) coupled to an Autospec (Micromass, Manchester, UK) MS.

The mass spectra were recorded with MS instrument at 70 eV.

4-3.9. Spectroscopy

4-3.9.1. NMR Spectroscopy

The 1H NMR spectra of the polysaccharide containing fractions F1, F1D and F1D-Sm

of S. polydactyla were recorded on a Bruker spectrometers (Bruker Biospin AG, Fallanden,

Switzerland) operating at 500 and 600 MHz for 1H. The proton nuclear magnetic resonance

(1H NMR) and

13C NMR spectra of the glucan (RbG) and experimentally sulfated glucan

(S1G) were recorded on a Bruker 400 spectrometer (Bruker Biospin AG, Fallanden,

Switzerland). The sulfated polysaccharides were converted into its sodium salt by passage

through a column (7 ml, Bio-Rad, Hercules, CA, USA) of Amberlite IR 120 (H+) followed

by neutralization using 50 mM NaOH solution, and all samples were deuterium-exchanged

by lyophilization with D2O (Cambridge Isotope Limited, Andover, USA) and then examined

as 1% solutions in D2O (99.96 atom % D). The chemical shifts are reported relative to an

internal acetone standard at 2.225 for 1H spectra and 31.55 for

13C spectra.


72 | P a g e

4-3.9.2. UV-VIS spectroscopy

UV-VIS spectra were recorded with a Shimadzu UV- 1800 spectrophotometer

(SHIMADZU, Japan).

4-3.9.3. Infra-Red spectroscopy

IR spectra were obtained on a Fourier transform (FT)-IR spectrophotometer (JASCO

FT/IR – 420 and FT/IR Spectrometer – Spectrum RX 1, PerkinElmer, USA) using KBr discs

containing finely powdered samples.

4-3.9.4. Mass spectrometry

EI mass spectra were recorded with Shimadzu QP5050A GLC/MS instrument at 70

eV. The characterization of partially methylated alditol acetates was done on the basis of

previously reported mass spectra (Carpita and Shea, 1988) and relative retention times (Ray

and Lahaye, 1995).

4-4. BIO – ASSAY

4-4.1. Cell culture and viruses

Vero (African green monkey kidney) cells were grown in Eagle's minimum essential

medium (MEM) supplemented with 5% fetal calf serum as described (Chattopadhyay, 2008;

Karmakar, 2010; Mandal, 2008; 2010). For maintenance medium (MM), the serum

concentration was reduced to 1.5%. Human diploid foreskin fibroblast cells were obtained

from Dr. G. Carballal (CEMIC, Buenos Aires, Argentina) and propagated in MEM

supplemented with 10% fetal calf serum.


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HSV-1 strain F and HSV-2 strain MS were used as reference strains. B2006 and Fields

are HSV-1 thymidine kinase negative (TK-) acyclovir-resistant strain obtained from Prof. Dr.

E. De Clercq (Rega Institute, Belgium). The syncytial variants of HSV-1 1C3-syn 13-8 and

1C3-syn 14-1 were obtained by serial passage in the presence of the mu/nu-carrageenan 1C3

obtained from the red seaweed Gigartina skottsbergii as previously described (Carlucci,

2002). Virus stocks were propagated and titrated by plaque formation in Vero cells.

In a separate experiment for sulfated rice bran glucans, Primary human foreskin

fibroblasts (HFFs) were cultivated in Eagle’s minimum essential medium (MEM) containing

7.5% (v/v) fetal calf serum (FCS). Human cytomegalovirus (HCMV, strain AD169-GFP);

(Marschall, 2000) and varicella zoster virus (VZV, vaccine strain Oka) were propagated in

HFFs and virus replication was quantified by automated green fluorescence protein (GFP)

fluorometry or plaque reduction assay, respectively (Marschall, 2000; Herget, 2004).

Epstein-Barr virus (EVB, strain B95-8) was used for infection of 293T cells (cultivated in

Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum) and virus replication

was quantified by applying a reporter-based replication assay (Rechter, 2006).

4-4.2. Anti viral assay

Anti viral activity was evaluated by a virus plaque reduction assay as described

(Chattopadhyay, 2007). Vero cell monolayers grown in 24-well plates were infected with

about 50 PFU/well in the absence or presence of various concentrations of the compounds.

After 1 h of adsorption at 37°C, residual inoculum was replaced by MM containing 0.7%

methylcellulose and the corresponding dose of each compound. Plaques were counted after 2

days of incubation at 37°C. The inhibitory concentration 50% (IC50) was calculated as the


74 | P a g e

compound concentration required to reduce virus plaques by 50%. All determinations were

performed twice and each in duplicate and results are expressed as mean value ± standard

deviation (SD).

4-4.3. Virucidal assay

Virucidal assay was performed by the procedure as described (Karmakar, 2010). A

virus suspension of HSV-1 (F) containing 4 x 106 PFU was incubated with an equal volume

of MM with or without various concentrations of the compounds for 2 h at 37°C. The

samples were then diluted in cold MM to determine residual infectivity by plaque formation.

The sample dilution effectively reduced the drug concentration to be incubated with the cells

at least 100-fold to assess that titer reduction was only due to cell-free virion inactivation.

The virucidal concentration 50% (VC50), defined as the concentration required to inactivate

virions by 50%, was then calculated.

4-4.4. Cytotoxicity assay

Vero cell viability was measured by the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-

diphenyl tetrazolium bromide; Sigma–Aldrich) method (Mandal, 2010). Confluent cultures

in 96-well plates were exposed to different concentrations of the polysaccharide, with three

wells for each concentration, using incubation conditions equivalent to those used in the

antiviral assays. Then 10 µl of MM containing MTT (final concentration 0.5 mg/ml) was

added to each well. After 2 h of incubation at 37°C, the supernatant was removed and 200 µl

of ethanol was added to each well to solubilize the formazan crystals. After vigorous shaking,

absorbance was measured in a microplate reader at 595 nm. The cytotoxic concentration 50%


75 | P a g e

(CC50) was calculated as the compound concentration required to reduce cell viability by

50%.

4-4.5. Assay for anti coagulant activity

Anticoagulant activity of the sulfated polysaccharides obtained from marine algae

was determined using the activated partial thromboplastine time (APTT) assay (Anderson,

1979) as described (Chattopadhyay, 2008) using heparin as the standard. Briefly, 30 µl of test

solution were added to 100 µl of pooled human plasma and 100 µl of APTT reagent (Wiener

lab, Argentina). The mixture was incubated for 1 min at 37°C. After the incubation, 70 µl of

CaCl2 0.025 M were added and the time to clot formation was recorded. Assays were

performed twice, in duplicate, and results are expressed as mean value ± S.D.

4-4.6. Influence of various treatment periods on the anti viral activity of the

compounds

Vero cells grown in 24-well plates were infected with 50 PFU of HSV-1 strain F

following different treatment conditions, as given below:

Adsorption: cells were exposed to HSV-1 in the presence of 10 µg/ml of the compound

(CiWE), and after 1 h at 4°C, both compound and unadsorbed virus were removed, the cells

were washed with cold PBS and overlaid with plaquing medium (MM containing 0.7%

methylcellulose).

Internalization: cells were infected with HSV-1 in the absence of compound and after 1 h of

adsorption at 4°C, the cells were washed twice with cold PBS and further incubated for 1 h at

37°C in MM containing 10 µg/ml of the compound. Then, cells were washed with cold PBS,


76 | P a g e

and 0.1 ml of citric buffer (40 mM citric acid, 10 mM KCl, 135 mM NaCl, pH 3.0) was

added to remove adsorbed noninternalized virus. After 1 min, cells were washed again with

PBS, and incubated with plaquing medium without compound.

After adsorption: cells were infected with HSV-1 in the absence of the compound and after

adsorption at 4°C, the unadsorbed virus was removed, the cells were washed two times with

cold PBS and further incubated with plaquing medium containing 10 µg/ml of the

compounds.

Always: the compound was present during HSV-1 adsorption at 4°C and in the plaquing

medium added after adsorption.

For all treatments, virus plaques were counted after 2 days of incubation at 37°C and results

were expressed as % inhibition for each treatment with respect to untreated infected cell

control.

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SUMMARY

The novel findings presented in this thesis are:

(i) A xylomannan with novel structural motif has been isolated from the marine red

algae Sebdenia polydactyla.

(ii) This polymer with an apparent molecular mass of 150 kDa contained a backbone

of α-(1→3)-linked-D-mannopyranosyl residues substituted at C6 with single stubs

of β-linked-D-xylopyranosyl residues. Sulfate groups, when present are located at

position 2 and / or 6 of the mannopyranosyl residues

(iii) The sulfated glucans generated from commercial preparation of rice bran (Oryza

sativa) contained α-(1→4)-linked-D-glucopyranosyl residues that are O-sulfated

at C6 and partly at C2 or C3.

(iv) These substances exerted strong inhibitory effect against different types of viruses

including herpes simplex virus (HSV) and human cytomegalovirus (HCMV).

(v) Sulfate content appeared to be an important hallmark of anti viral activity.

(vi) The main mode of action of these drug candidates was directed to viral entry.

(vii) Generally polysaccharides from higher plants do not contain sulfate group. This is

the first study where a neutral glycan from rice bran was chemically sulfated and

their structures and anti HCMV activity were studied.

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List of publications

1. Ghosh, T., Pujol, C. A., Damonte, E. B., Sinha, S. and Ray, B. 2009. Sulfated xylomannans from

the red seaweed Sebdenia polydactyla: structural features, chemical modification and antiviral

activity. Antiviral Chemistry & Chemotherapy, 19, 235-242.

2. Ghosh. T., Auerochs, S., Saha, S., Ray, B. and Marschall, M. 2010. Anti-cytomegalovirus activity

of sulfated glucans generated from a commercial preparation of rice bran. Antiviral Chemistry and

Chemotherapy, 21, 85-95.

3. Ghosh, T., Chattopadhyay, K., Marschall, M., Karmakar, P., Mandal. P. and Ray, B. 2009. Focus on

antivirally active sulfated polysaccharides: from structure-activity analysis to clinical evaluation.

Glycobiology, 19(1), 2-15.

4. Chattopadhyay, N1., Ghosh, T

1., Sinha, S., Chattopadhyay, K.,

Karmakar, P. and Ray, B. 2010.

Polysaccharides from Turbinaria conoides: Structural features and antioxidant capacity. Food

Chemistry, 118, 823-829.

5. Bandyopadhyay, S. S., Navid, M. H., Ghosh, T., Schnitzler, P. and Ray, B. 2011. Structural features

and in vitro antiviral activities of sulfated polysaccharides from Sphacelaria indica. Phytochemistry,

72, 276-283.

6. Sinha, S., Astani, A., Ghosh, T., Schnitzler, P. and Ray, B. 2010. Polysaccharides from Sargassum

tenerrimum: Structural features, chemical modification and anti-viral activity. Phytochemistry, 71,

235-242.

7. Karmakar, P., Ghosh, T., Sinha, S., Saha, S., Mandal, P., Ghosal, P. K. and Ray, B. 2009.

Polysaccharides from the brown seaweed Padina tetrastromatica: Characterization of a sulfated

fucan. Carbohydrate Polymers, 78, 416–421.

8. Chattopadhyay, K., Ghosh, T., Pujol, C. A., Carlucci, M. J., Damonte, E. B. and Ray, B. 2008.

Polysaccharides from Gracilaria corticata: Sulfation, chemical characterization and anti-HSV

activities. International Journal of Biological Macromolecules, 43, 346–351.

9. Karmakar, P., Pujol, C. A., Damonte, E. B., Ghosh, T. and Ray, B. 2010. Polysaccharides from

Padina tetrastromatica: Structural features, chemical modification and antiviral activity.

Carbohydrate Polymers, 80, 514-521.

10. Mandal, P., Pujol, C. A., Damonte, E. B., Ghosh, T. and Ray, B. 2010. Xylans from Scinaia hatei:

Structural features, sulfation and anti-HSV activity. International Journal of Biological

Macromolecules, 46, 173-178.

100 | P a g e

Book chapter: One (01)

1. Chattopadhyay, K., Ghosh, T., Karmakar, P., Mandal, P., Ghosh, P. and Ray, B. 2009.

Ethnopharmacological Uses, Chemical Constituents and Biological Activities of Sesame: A Review.

In: Recent Progress in Medicinal Plants, 23, 91-113, Studium Press LLC, USA.

Documents

POLYSACCHARIDES FROM Sebdenia polydactyla AND Oryza …shodhganga.inflibnet.ac.in/bitstream/10603/21972/2/2 thesis.pdf · Polysaccharides are carbohydrates that form the bulk of all