POLYSACCHARIDES FROM Turbinaria conoides AND...

THESIS SUBMITTED FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY (SCIENCE) IN CHEMISTRY

OF THE UNIVERSITY OF BURDWAN

2013

POLYSACCHARIDES FROM

Turbinaria conoides AND Adhatoda vasica :

STRUCTURAL FEATURES AND BIOLOGICAL

ACTIVITIES

Nabanita Chattopadhyay, M. Sc.

NATURAL PRODUCTS LABORATORY

DEPARTMENT OF CHEMISTRY

THE UNIVERSITY OF BURDWAN

WEST BENGAL, INDIA

Dedicated to my beloved parentsDedicated to my beloved parentsDedicated to my beloved parentsDedicated to my beloved parents

THE UNIVERSITY OF BURDWAN

TO WHOM IT MAY CONCERN This is to certify that the thesis “POLYSACCHARIDES FROM Turbinaria

conoides AND Adhatoda vasica: STRUCTURAL FEATURES AND

BIOLOGICAL ACTIVITIES” is the result of work done by Nabanita

Chattopadhyay, M.Sc., who has registered her name in The University of

Burdwan on 19-03-2007 for the award of Doctor of Philosophy (Science) in

Chemistry under my supervision and Guidance. This work neither in part nor

whole has been submitted for any degree by Nabanita Chattopadhyay or other. It is

also certified that she has successfully completed Ph.D. course work framed by the

Department of Chemistry, following the guidelines of The University of Burdwan,

and that she has delivered one seminar lecture on 02-08-2012 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)

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…………………..

ACKNOWLEDGEMENT

My journey at the University of Burdwan would not have become fruitful without the advice

and assistance of some special persons. Let me at the outset express my heartfelt gratitude to my

revered teacher and supervisor Dr. Bimalendu Ray, Associate Professor, Department of

Chemistry, The University of Burdwan, for his continuous supportive guidance, untiring help

and encouragement during the course of my work. I am very much thankful to him for his active

participation in all of my publications related to this thesis.

I would like to express my sincere gratitude to Prof. Pradyot Ghosal, Prof. Subrata Laskar,

and Dr. A. K. Ghosh, the present head of the Department of Chemistry, The University of

Burdwan, for their generous help, constant encouragement.

I record my indebtedness to Prof. Gabriella Nosál’ova, Department of Pharmacology,

Jessenius Faculty of Medicine, Comenius University, Martin, Slovakia, who helped me with

Bioassay. I am thankful 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, Dr. Tuhin Ghosh, Mr. Sudipta

Saha, Mr. Udipta Ranjan Chatterjee, Mr. Shruti Sourav Bandyopadhyay, Mr. Sujay Majee, Mrs.

Paramita Karmakar, Ms.Sharmistha Sinha, Ms. Debjani Ghosh, Ms. Kanika Ghosh deserve

special mention.

My sincere thanks are cordially extended to Mrs. Tapasi Ray for her enthusiastic

encouragement and cooperation. The infrastructural facilities from The University of Burdwan

are gratefully acknowledged.

Finally, my special acknowledgements are owed to my parents, my sister, brother-in-law,

brother, and last but not the least my husband for their help, constant inspiration, encouragement

in course of my research activity.

Department of Chemistry,

The University of Burdwan,

Burdwan, 713104, India. (Nabanita Chattopadhyay)

CONTENTS

1. POLYSACCHARIDES FROM NATURAL SOURCES: BIOLOGICAL ROLES, INDUSTRIAL USES AND PHARMACOLOGICAL ACTIVITIES

1-1. INTRODUCTION

1-2. BIOLOGICAL ROLES

1-3. INDUSTRIAL USES

1-4. PHARMACOLOGICAL ACTIVITIES

1-4.1. Anticoagulant activity

• Structure-activity relationships

• Mechanism of action

1-4.2. Anticomplementary activity


1-4.3. Antioxidative activity


1-4.4. Antitumor activity



1-4.5. Antitussive activity



1-4.6. Antiviral activity



1-4.7. Potential clinical applications

10

11

12

14

15

16

18

19

19

20

21

22

9

8

7

6

5

4

3

3

2

AIMS

2. STRUCTURAL FEATURES AND ANTIOXIDATIVE ACTIVITIES OF CARBOHYDRATE POLYMERS FROM THE BROWN SEAWEED Turbinaria conoides

2-1. INTRODUCTION

2-2. CHEMICAL CHARACTERIZATION OF POLYSACCHARIDES FROM Turbinaria conoides

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

analysis

2-2.2. Isolation and sugar composition of polysaccharide fractions

2-2.3. Purification of the fucoidans by chromatography

2-2.4. Molecular mass

2-2.5. Linkage analysis

2-2.6. NMR spectroscopy

2-2.7. Structural features of the glucan and alginic acid rich fractions

2-3. PHARMACOLOGICAL ACTIVITIES OF POLYSACCHARIDES FROM Turbinaria conoides


• Ferric ion reducing/antioxidant power (FRAP) assay

• Scavenging effect on 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radicals

2-4. CONCLUSIONS

24

27

28

28

30

32

33

33

36

37

38

38

38

39

42

3. STRUCTURAL FEATURES AND ANTITUSSIVE ACTIVITIES OF A CARBOHYDRATE POLYMER FROM Adhatoda vasica

3-1. INTRODUCTION

3-2. CHEMICAL CHARACTERIZATION OF THE PECTIC ARABINOGALACTAN FROM Adhatoda vasica

3-2.1. Isolation and chemical composition

3-2.2. Size exclusion chromatography (SEC)


3-3. ANTITUSSIVE ACTIVITIES OF THE PECTIC ARABINOGALACTAN FROM Adhatoda vasica

3-3.1. Assessment on chemically induced cough and airways defense reflexes

3-3.2. Mechanism of action

3-4. CONCLUSIONS

4. MATERIALS AND METHODS

4-1. PLANT MATERIALS AND EXTRACTION OF POLYSACCHARIDES

4-1.1. Isolation of sulfated fucan, alginic acid and glucan from Turbinaria conoides by sequential extraction with inorganic solvents

• Plant material and preliminary treatments

• Preparation of depigmented algal powder (DAP)

• Extraction with acid

• Extraction with alkali

• Extraction with water

4-1.2. Isolation of polysaccharides from Adhatoda vasica

• Plant material and preliminary treatments

• Extraction of polysaccharides

44

46

46

47

48

50

50

51

52

55

55

55

55

55

55

55

56

56

56

• Isolation of arabinogalactan protein (AGP) with β-glucosyl Yariv reagent

4-2. ANALYTICAL METHODS

4-2.1. General 4-2.2. Sugar analysis

4-2.3. Protein estimation

4-2.4. Sulfate estimation

• Turbidometric method

• Spectroscopic method

4-2.5. Desulfation 4-2.6. Linkage analysis

4-2.7. Chromatography

• Thin layer chromatography (TLC)

• Size exclusion chromatography (SEC)

• Anion Exchange Chromatography (AEC)

• Gas chromatography (GC)

4-2.8. Spectroscopy

• NMR Spectroscopy

• UV-VIS spectroscopy

• Infra-Red spectroscopy

• Mass spectrometry

4-3. BIOASSAY

4-3.1. FRAP assay

4-3.2. Scavenging capability for 1,1-diphenyl-2-picrylhydrazyl

(DPPH) radicals

56

56

56

57

57

58

58

58

58

58

59

59

59

60

60

60

60

61

61

61

61

61

61

4-3.3. Antitussive activity of the arabinogalactan from A. vasica

• Animals

• Assessment of chemically induced cough and airways defense reflexes

• Statistics

REFERENCES SUMMARY LIST OF PUBLICATIONS

62

62

62

62

64

80

81

1

POLYSACCHARIDES FROM NATURAL SOURCES: BIOLOGICAL ROLES,

INDUSTRIAL USES AND PHARMACOLOGICAL

ACTIVITIES

0

100

200

1-1. INTRODUCTION

Carbohydrates are the fuel of life, being the main source of energy for living organisms and

the central pathway of energy sto

through which the energy of the sun is harnessed and converted into a

by living organisms. According to rough estimates, carbohydrate

annually regrowing biomass of about 200 billion tons

man, the rest decays and recycles along natural

Polysaccharides, proposed as the first biopolymers to have formed on Earth (Tolstoguzov,

2004), are a major group

macromolecule can be defined in terms of the composition

configuration and sequence of its constituent sugars as well as the presence of any non

residues and their positions. Polysaccharides also have secondary, tertiary and quat

structures, which depend upon the repeating sequence in primary structure, the confor

glycosidic linkages and aggregation of polymer chain by various non

two decades the mechanism behind the biological

uses and therapeutic applications

documenting the developments

Fig. 1.1. Carbohydrate is a primary component of renewable biomass

Bil

lio

n t

on

s

Carbohydrate

Non-Carbohydrate


the central pathway of energy storage and supply for most cells. They are the major products

through which the energy of the sun is harnessed and converted into a form that can be utilized

by living organisms. According to rough estimates, carbohydrates represent roughly 95% of the

annually regrowing biomass of about 200 billion tons (Fig. 1.1); of these only 3% are used by

the rest decays and recycles along natural pathways (Lichtenthaler &

proposed as the first biopolymers to have formed on Earth (Tolstoguzov,

2004), are a major group in carbohydrate chemistry. The primary structure of this

macromolecule can be defined in terms of the composition, glycosidic linkage, anomeric

and sequence of its constituent sugars as well as the presence of any non

. Polysaccharides also have secondary, tertiary and quat

upon the repeating sequence in primary structure, the confor

linkages and aggregation of polymer chain by various non-covalent bonds.

mechanism behind the biological roles of carbohydrate polymers,

and therapeutic applications have increased tremendously. This

the developments of these macromolecules in these fields.

1. Carbohydrate is a primary component of renewable biomass

2


They are the major products

form that can be utilized

represent roughly 95% of the

; of these only 3% are used by

Mondel, 1997).

proposed as the first biopolymers to have formed on Earth (Tolstoguzov,

The primary structure of this

, glycosidic linkage, anomeric

and sequence of its constituent sugars as well as the presence of any non-sugar

. Polysaccharides also have secondary, tertiary and quaternary

upon the repeating sequence in primary structure, the conformation of

covalent bonds. In the last

polymers, their industrial

This chapter aims at

1. Carbohydrate is a primary component of renewable biomass.

3

1-2. BIOLOGICAL ROLES

Carbohydrate polymers occur in almost all living organism and serve diverse functions in the

living material in which they are endogenous. The function of cellulose, the most abundant

naturally occurring substance, as structural support in plant is well established. But, in animals,

they rarely serve such purposes as structural support. However, the special physical texture and

the hydrophilic character are responsible for their multi-various roles. Cell walls of many

bacteria contain polysaccharides, which are responsible for their protective coatings and

serologic specificity (Holst, 1999). Some bacterial polysaccharides are highly antigenic having

endotoxin properties. Other natural macromolecules, which are not composed entirely of sugar

units, contain blocks of monosaccharide units as part of the molecular structure, and contribute

extensively to the production and maintenance of living tissues of animals. The blood-group

polysaccharide constitutes a group of glycoproteins in which arrangement of monosaccharide

residues in carbohydrate subunits controls the blood-group specificity to the overall molecule

(Feizi, 2000). Immunoglobulins are a group of glycoprotein that has antibody activity (van de

Perre, 2003). Transferrin is a glycoprotein, which forms complexes with iron and is responsible

for transporting iron from the storage form in tissues, especially in liver to the metabolically

functioning iron in hemoglobin (Kawabata et al., 2000). Glycosaminoglycans are amongst the

essential building blocks of the macromolecular framework of connective and other tissues

(Taylor & Gallo, 2006). Hyaluronic acid appears to act, on account of its viscosity in solution, as

a lubricant, shock-absorbing gel in limb joints (Kogan et al., 2007).

1-3. INDUSTRIAL USES

Interest in polysaccharides, however, is not purely because of its biological activities but

rather is prompted by their great utility as a raw material in many industries such as food,

pharmaceutical, etc. Water-soluble polysaccharides have enormous use in food industries. Their

non-toxicity, wide availability, and low cost make them suitable for food industries. Also, these

macromolecules are chosen for their added values, as for example for their low calorie intake.

Importantly, they are mostly used as additive to improve or control food properties. Notably,

their applications largely depend on the properties, they impart to solution and gels. The different

families of polysaccharides and their uses in food industry are as follows: (i) anionic exudates

4

polysaccharides such as gum arabic used as an emulsifying agent in the precandy jellies such as

jujubes, fruit gums, fruit pastille, gum drops and cough drops & stabilizing agent as angel kisses,

marshmallows, soft caramels, nougats and meringues (Kubal & Gralen, 1948; Meer, 1980), gum

tragacanth and karaya mainly used as thickener and emulsifier in sauces, salad dressings and

confectionery lozenges (Whistler, 1993), (ii) anionic seaweed polysaccharides including agars

used as gelling agent in wine and fruit juice (Tseng, 1946; Selby & Whistler, 1993), alginates as

gelling agent in making jam, jellies, fruit fillings etc & as stabilizing agent in beer, pulp, fruit

drinks without forming haze and low fat substitutes and carrageenans used as stabilizing agent in

dairy products such as milk chocolate, yogurts and egg nog mixes, ice-cream etc (Painter, 1983;

Piculell, 1995), and also in non-dairy food product, such as instant products, jellies, pet foods,

sauces (Therkelsen,1993; Imeson, 2000), (iii) microbial polysaccharides such as gellan gum &

xanthan gum (extensively used in the food industry as stabilizing, thickening, gelling agent), (iv)

non-ionic seed polysaccharides including guar gum (an economical thickener and stabilizer in ice

creams, sherbets and related products, breads, cakes and donuts & other dairy products),

tamarind seed gum, locust bean gum and (v) pectin, mainly used as gelling agent but also as

stabilizing, thickening and suspending agent (May, 1990; Rolin & DeVries, 1990; Pilnik, 1992).

Polysaccharides commonly used in non food industry are (i) anionic exudates polysaccharides

such as gum tragacanth and karaya (Whistler, 1993), (ii) anionic seaweed polysaccharides such

as agars (Tseng, 1946; Selby & Whistler, 1993) and carrageenans (Painter, 1983; Therkelsen,

1993; Piculell, 1995; Imeson, 2000), (iii) microbial polysaccharides such as dextran (DeBelder,

1993), and xanthan.

The industrial applications of polysaccharides depend on their unique properties, often at a

cost, much lower than the synthetic polymers. They can produce gels of different strength and

stability under controlled condition. Also, they can act as good emulsifier by their interfacial

binding properties. Polysaccharides are useful to supply required consistency by controlling the

moisture. They are important as additives in relatively low proportions, as for example as

thickening, stabilizing agents.

1-4. PHARMACOLOGICAL ACTIVITIES

Recognition of the pharmacological properties of myriad polysaccharides has fueled the

current focus on the search for new drug candidates. Besides their well-attested anticoagulant

5

and antithrombotic activities, they can act on complement systems, have tumoricidal, antitussive

and antioxidative properties, and can protect cells from viral infection (Yamada & Knutsen,

1996; Franz et al., 2000; Paulsen & Samuelsen, 2001; Inngjerdingen et al., 2006; Balzarini &

Van Damme, 2007; Mantovani et al., 2008; Ghosh et al., 2009a; Baek et al., 2010; Sinha et al.,

2011a, 2011b; Bandyopadhyay et al., 2012; Thakur et al., 2012).

The next part of this chapter, which summarizes experimental evidences indicating that

polysaccharides might play increasingly important roles in the prevention of diseases in the near

future, is divided into six parts according to their pharmacological activities. Further

classification within each activity is made by structural type.

1-4.1. Anticoagulant activity

Heparin is a heterogeneous group of straight-chain anionic mucopolysaccharides, called

glycosaminoglycans, having anticoagulant properties (Linhardt & Toida, 1997; Gunay &

Linhardt, 1999; Alban & Franz, 2001; Lee et al., 2008; Fan et al., 2011). This linear

polysaccharide consists of simple disaccharides repeats of alternating unit of (1→4)-linked

uronic acid and glucosamine, made complex by variation of substitutions with O- and N- sulfo

and N- acetyl groups, as well as by epimerization of the uronic acid (Mulloy & Linhardt, 2001;

Feyerabend et al., 2006). This compound has very much higher specific activity as an

anticoagulant than other sulfated polysaccharides and this potency depends on a specific

pentasaccharide sequence (Berteau & Mulloy, 2003; Mulloy, 2005). However, because of

several side effects (Pereira et al., 2002; Athukorala et al., 2007) and limited availability of

heparin, there is considerable interest to obtain safer anticoagulants (heparinoids) from other

natural sources (Alban et al., 2002). In fact, the first report of the heparinoid activity of high-

molecular-weight polysaccharides appeared almost 55 years ago (Springer et al., 1957).

Recently, a number of sulfated polysaccharides from marine sources have emerged as an

important class of compound having anticoagulant effects. Among them, fucoidans are well

known for their heparinoid activity (Nishino & Nagumo, 1991, 1992; Chauvet et al., 1999;

Chevolot et al., 1999; Pereira et al., 1999; Berteau & Mulloy, 2003; Silva et al., 2005; Cumashi

et al., 2007; Chandia & Matsuhiro, 2008). Based on structural features fucoidans are of three

major types: (i) a polysaccharide based on L-fucose with mainly α-(1→3)-glycosidic bonds and

sulfate groups at position 4 (Patankar et al., 1993) (Fig. 1.2), (ii) fucoidan possessing large

proportions of both α-(1→3)-and α-(1→4)-glycosidic bonds (Chevolot et al., 1999, 2001;

6

Daniel et al., 1999, 2001) and (iii) macromolecule is composed primarily of α-(1→2)- and α-

(1→3)-linked fucopyranosyl residues with sulfate groups at position 4 and 2 (Karmakar et al.,

2009).

Besides fucoidan, sulfated galactan (Farias et al., 2000; Matsubara et al., 2001; Farias et al.,

2008; Glauser et al., 2009) and several other sulfated heteropolysaccharides having anticoagulant

activity has also been indicated (Rojers et al., 1990; Guven et al., 1991; Maeda et al., 1991;

Potin et al., 1992). Notably, the anticoagulant activity of some of these compounds such as

rhamnan sulfate, a macromolecule isolated from Monostroma spp., is six folds higher than that

of standard heparin (Maeda et al., 1991). Furthermore structurally well defined sulfated

polysaccharides produced by chemical sulfation also found to have antithrombotics properties

(Alban & Franz, 2001; Alban et al., 2002).

Structure-activity relationships. First, it has been observed that for polysaccharides with the

same structure, the anticoagulant activity depends on the ratio of sulfate group to total sugar

residues of the polysaccharides (Nishino & Nagumo, 1991, 1992). The higher the ratio is, the

OH3C

O

O

O

OR1

R2O

OR3

H3C

O

OH3C

OOR1

R2O

R2O

OH3C

OOR1

R2O

OH3COR1

R2O

R1 = H or SO3- or COCH3

R2 = H or SO3-

R3 = OH3C

HO

O

OR2

R2O

Fig. 1.2. The quasi-repeat unit identified in 1→3-linked fucoidan from Chorda filum (Chizhov et al.,

1999). Other substituents, such as O-acetyl, and branches are present in all these fucoidans and add

considerably to their heterogeneity.

7

better are the chances of exhibiting higher anticoagulant activity. Nishino and colleagues have

found that higher the content of fucose and sulfate groups present, higher is the anticoagulant

activity in native fucoidans from Ecklonia kurome (Nishino et al., 1989; Nishino & Nagumo,

1991, 1992). But it is always not a rule. For example, it has been observed that although the ratio

(0.75) of sulfate to sugars of a fucoidan fraction isolated from commercial Fucus vesiculosus was

higher than those (0.31-0.68) of other fractions, but the former polysaccharide preparation was,

nevertheless, inactive (Nishino et al., 1994). These results clearly demonstrate that not only the

sulfate group but also structural features are responsible for the anticoagulant activity of sulfated

polysaccharides. Second, the location of sulfate group and/or the glycosidic linkage position

affect the activity, as indicated by the comparison between the inactive 3-sulfated 4-linked and

the active 2-sulfated 3-linked α-L-galactans (Mourao, 2004). Third, the occurrence of 2, 4-di-

sulfated units has an amplifying effect on the antithrombin-mediated anticoagulant activity of 3-

linked α-L-fucoidan. This is not merely a consequence of increased charge density. The

anticoagulant activity increases ~38-fold from a 2-sulfated 3-linked α-L-fucoidan to a 2, 4-

disulfated α-L-fucoidan, even though their sulfate content increases only ~1.8-fold (Pomin &

Mourao, 2008). Fourth, the anticoagulant activity of a particular family of polysaccharide also

depends upon their molecular mass. The native fucoidan (MW 320,000 Da) from Lessonia

vadosa have good anticoagulant activity, whereas the radical depolymerized fraction (MW

32,000 Da) exhibits weak anticoagulant activity (Chandia & Matsuhiro, 2008). Finally, the

structures of sulfated polysaccharides vary based on their algal source species to species and give

rise to variation in the degree of anticoagulation action (Chevolot et al., 1999; Pereira et al.,

1999; Boisson-Vidal et al., 2000; Pomin et al., 2005).

Mechanism of action. The biological activities of heparin are due to its binding to cellular

proteins modulating their activities. This interaction is often very specific (Gunay & Linhardt,

1999; Mulloy, 2005), e.g., heparin’s anticoagulant activity mainly results from binding

antithrombin III (ATIII, a serine protease inhibitor of thrombin and other coagulation proteases)

at a discrete pentasaccharide sequence (Fig. 1.3). Sulfated polysaccharides contain several types

of functional groups such as sulfate esters, carboxyls, hydroxyls and, therefore, able to interact

with cellular proteins via: (i) hydrogen bond, (ii) electrostatic interaction, (iii) hydrophobic

interactions and (iv) van deer Waals forces (Hileman et al., 1998; Quiocho & Vyas, 1999).

Electrostatic interaction can occur between locally positively charged patches on the protein

8

(under normal physiological pH) and sulfate groups of heparinoids (reviewed by Gunay &

Linhardt, 1999). The anionic substituents (sulfate and carboxylate groups) in the saccharide

backbone of heparin and some heparinoids have different spatial arrangements, which stem from

a various degree of chirality, different regioisomers, conformers and secondary structure in

carbohydrate backbone. Ease in re-orientation of charged groups, owing to the flexible

saccharide backbone, further assists in such interactions. Heparinoids can also form hydrogen

bonds with the polar amino acids of binding proteins. The residues involved in ionic and

hydrogen bonding interactions are usually spatially arranged either on the surface or on the

binding pocket of the proteins. Substantial hydrophobic contributions to binding may result in

the interaction of heparinoids having hydrophobic character (i.e. fucoidan) making these

effectively potent agents (Gunay & Linhardt, 1999).

1-4.2. Anticomplementary activity

The complement system is part of the innate immune system and consists of a group of serum

proteins which are activated in a cascade mechanism (Samuelsen et al., 1996). This system is

important in initiating inflammation, and its activation might result in the cellular co-operation,

immunopotentiation and regulation of cyclical antibody production. This activation may be

beneficial to the host, but it may also be harmful by damaging host cells and tissues. Many

naturally occurring polysaccharides from plant, bacteria, lichen, fungi have effects on the human

immune system. These compounds include pectin, pectic arabinogalactan (AG), and other acidic

heteroglycans. In addition, several neutral polysaccharides such as glucan (Tomoda et al., 1994a,

1994b, Olafsdottir et al., 1999a), arabinan (Yamada et al., 1989b), arabinoglucan (Yamada et

al., 1989b), galactan (Yamada et al., 1989a), rhamnopyranosylgalactofuranan (Olafsdottir et

al.,1999b), galactomannan (Latge et al., 1994), arabinoxylan (Hromadkova et al., 2012), and

other hetero polysaccharides (Tomoda et al., 1994b; Zhao et al., 1994) with anticomplementary

O

CH2SO3

OH

O

O

O

(Ac or SO3) HN

CO2

OH

OH

O

O

CH2OSO3

OSO3

NHSO3

O O

O

CO2OH

OSO3

O

CH2OSO3

OH

NHSO3

O

Fig. 1.3. Discrete pentasaccharide sequence of Heparin (Mulloy & Linhardt, 2001).

9

activity have been isolated from medicinal plants or released from pectic polysaccharides during

isolation. Finally, carbohydrate polymers that are not composed entirely of monosaccharide

residues can also activate complement system. For example, the highly branched protein-bound

polysaccharide (~ 1000-2000 kDa), containing terminal, 1,3-, 1,4-, 1,2,6-, 1,3,6-linked glucose;

1,6-, 1,2,6-, 1,3,4-, 1,4,6-linked galactose; 1,5-, 1,3,5-linked arabinose, terminal and 1,2,5-linked

rhamnose residues and 6.55% of protein from Eucommia showed potent anticomplementary

activity (Zhu et al., 2009).

Structure-activity relationships. Because of great chemical variety, it is very difficult to

correlate between structure and activity. However, on the basis of accumulated data the

following conclusions could be made for pectic polysaccharides: (i) the “smooth” region i.e., the

region which consists of polygalacturonide moiety (Voragen et al., 1995) have none or only

weak activities, (ii) esterification, acetylation or uronic acid content of the polymers do not affect

activity (Kiyohara et al., 1989, 1993; Yamada et al., 1989a, 1992; Hirano et al., 1994; Tomoda et

al., 1994a; Samuelsen et al., 1996, 1998; Shin et al.,1998; Yu et al., 1998; Sakurai et al., 1999),

(iii) the hairy region (Voragen et al., 1995) of the pectin in particular the carbohydrate side

chains that attached to the hairy region, might be essential for the expression of this activity

(Kiyohara et al., 1989, 1993; Yamada et al., 1992; Samuelsen et al., 1996, 1998), (iv)

polyanionic smooth regions in the pectin might modulate the mode of complement activation

induced by the hairy region (Kiyohara et al., 1989), (v) a high degree of branching may be

important for the activity and arabinose attached terminally to the galactan side chains reduces

the activity, while arabinose side chain attached directly to C3 of the galacturonic acid residues

increases the activity (Samuelsen et al., 1996), (vi) molecular weight also plays an important role

in exhibiting this activity. For example, an arabinogalactan from Atractylodes lancea DC having

molecular weight (74,000) showed a strong reactivity, whereas other arabinogalactans

(molecular weight 3,100 & 16,000) from the same source did not show the reactivity (Yu et al.,

1998). A careful study of the monomerization and dimerization of the rhamnogalacturonan II

(RG II, a type of pectic polysaccharide having very complex structure) isolated from the leaves

of Panax ginseng clearly indicated that the RG II dimers cross-linked by borate diesters strongly

contribute to the activity (Shin et al., 1998). The higher the proportion of dimer the higher is the

activity. Among branched α-D-glucan, polymers with higher degree of branching have higher

potency (Tomoda et al., 1994a; 1994b), but for linear glucan the presence of α-(1→3)-linkages

10

is essential (Olafsdottir et al., 1999a). Besides, the higher the molecular mass of the

macromolecule, the better is its potency (Olafsdottir et al., 1999a).

These facts clearly demonstrate that besides primary structure some other features are

responsible for the biological activity of pectin. An attractive concept is that polymers having

comb like conformation exhibit anticomplementary activity. Hairy regions of the pectic

substances are characterized by a linear backbone with side chain and hence possess a comb-type

structure. Arabinogalactan, a branched polymer, obtained from various sources showed

anticomplementary activity (Wagner & Jordan, 1988; Kiyohara et al., 1993; Samuelsen et al.,

1998; Yu et al., 1998). So it seems that a comb-like structure with a linear backbone and

branched side-chains may be prerequisite for this activity.


A biological antioxidant has been defined as “any substance that, when present at low

concentrations compared to those of an oxidizable substrate, significantly delays or prevents

oxidation of that substrate” (Halliwell & Gutteridge, 1995). Although oxidation reactions are

crucial for life, they can also be damaging and can cause many diseases like cancer (Paz-Elizur

et al., 2008), liver disease (Preedy et al., 1998), Alzheimer’s disease (Moreira et al., 2005), aging

(Liu & Mori, 2006), arthritis (Colak, 2008), inflammation (Mukherjee et al., 2007), diabetes

(Naito et al., 2006; Jain, 2006), Parkinson’s disease (Beal, 2003; Chaturvedi et al., 2008),

atherosclerosis (Heinecke, 1997), and AIDS (Sepulveda & Watson, 2002). Natural compounds

offer interesting pharmacological properties for their use as antioxidative agent. In recent years, a

number of polysaccharides containing fractions isolated from various sources as for example,

marine algae (Ruperez et al., 2002; Rocha de Souza et al., 2007; Wang et al., 2008, 2009a),

plants (Aguirre et al., 2009; Yang et al., 2011), and even some enzymatic extracts (Je et al.,

2009) possess antioxidative activity. Some of them, in particular the sulfated polysaccharides

from marine algae such as fucoidan (Zhao et al., 2005; Rocha de Souza et al., 2007; Wang et al.,

2008, 2009b; Wang et al., 2010), sulfated galactan (Barahona et al., 2011), sulfated

polysaccharide fractions containing galactose and xylose residues as constituent sugar, and

rhamnose-rich polysaccharide fractions showed considerable antioxidative properties (Hu et al.,

2010; Yang et al., 2011).

Polysaccharides from higher plants such as arabinogalactan (Fig. 1.4) also showed

antioxidative activity (Chatterjee et al., 2011; Sinha et al., 2011a). The highly branched

11

arabinogalactan isolated from Indian medicinal plant Eugenia jambolana is esterified with

phenolic acid and possess potent dose dependent antioxidative activity (Bandyopadhyay et al.,

2012).

In addition, chemically modified macromolecules such as acetylated and benzoylated ulvans

from Ulva pertusa (Chlorophyta) also possess antioxidative property. Potency of some of these

compounds is higher than the others (Qi et al., 2006).

Structure-activity relationships. The antioxidative property of polysaccharides depends upon a

number of parameters including molecular mass (Qi et al., 2005). In case of sulfated

polysaccharides, potency depends upon their sulfate content (Qi et al., 2005; Hu et al., 2010).

For example, sulfated polysaccharides from Undaria pinnitafida had stronger antioxidant

abilities than their de-sulfated derivatives (Wang et al., 2008). Recent study showed that the

position of sulfate group is another important parameter. The increased hydrophobic character of

Fig. 1.4. A model showing the structural features of an arabinogalactan from the Indian medicinal plant Eugenia jambolana (Bandyopadhyay et al., 2012).

12

the macromolecule also leads to higher potency (Qi et al., 2006). Finally, the structure of

polysaccharide also plays an important role in antioxidative property.

1-4.4. Antitumor activity

Polysaccharides also possess antitumor activity (Table 1.1). These macromolecules usually

have low toxicity and few side effects, which is essential for immunotherapy against cancer.

Class of carbohydrate polymer

Sources Type of source Reference

ββββ-D glucan Lentinus edodes Fungi Chihara et al., 1970; Ooi & Liu, 2000

ββββ-D glucan Schizophyllum commune Fungi Ooi & Liu, 2000

ββββ-D glucan Ganoderma lucidum Fungi Boh et al., 2007; Chan et al., 2009

ββββ-glucan Thamnolia vermicularis Lichen Olafsdottir et al., 2003

Protein-bound β-glucan Coriolus versicolor Fungi Ooi & Liu, 2000, Hobbs, 2004

Glucomannan Agaricus blazei Fungi Mizuno et al., 1999; Tsuchida et al., 2001

Galactomannans coffee Simões et al., 2010

Glucurono-gluco-galactomannan

Aureobasidium pullulans Bacteria Kataoka-Shirasugi et al., 1994

Fucogalactomannan Grifola frondosa Fungi Zhuang et al., 1994

Acidic glycoprotein Flammulina velutipes (W. Curt.: Fr.) Singer

Fungi Maruyama & Ikekawa, 2007

Amongst these glycans, the branched β-D-glucans are the most useful antitumor compounds.

The major structural feature of these active biopolymers is shown in (Fig. 1.5). These

macromolecules differ from each other by their chain length and branching (Stone & Clarke,

1992).

Table 1.1. Antitumor activity of different carbohydrate polymers from various natural sources

13

Fig. 1.5. Molecular core of β-D glucan.

OHO

OOH

O

OH

OHO

OH

OHO

OHO

OH

O*

O

HO

HO

HO

O

OH

n

However, the branches in the glycosidic chain are highly variable and the two main branching

are (1→4) or (1→6) glycosidic chains. Some naturally occurring β-glucans mainly Lentinans,

Schizophyllan, PSK (Krestin) are of particular clinical interest. Lentinan, produced from Shiitake

mushroom, Lentinus edodes, is a β-(1→3)-D-glucan having two β-(1→6)-glucopyranoside

branches for every five β-(1→3)-glucopyranoside linear linkages (Sasaki & Takasuka, 1976;

Saito et al., 1977, 1979). Schizophyllan from the mushroom of Schizophyllum commune consists

of β-(1→3)-D-glucan having one β-(1→6)-D-glucopyranoside branching for every three β-

(1→3)-D-glucopyranoside in the main chain. On the otherhand PSK has a basic β-glucan

structure with β-(1→6)-glucopyranosyl side chains every fourth glucose unit (Fisher & Yang,

2002). The PSK branched structure includes (1→3), or (1→4) and (1→6) bonds, and branches at

3- and 6-positions in a proportion of one per several residual groups of 1→4 bonds (Wasser,

2002; Boh et al., 2007).

Among acidic polysaccharides of natural origin the alginate (Ye et al., 2008), carrageenan

(Zhou et al., 2004), fucoidan (Usui, 1980; Maruyama et al., 2006; Synytsya et al., 2010), pectin

(Edenharder et al., 1995; Hensel & Meier, 1999; Inngjerdingen et al., 2007) etc. have been

examined for their antitumor activities. Alginic acid is one of the best-studied polymers with

pronounced activities against various tumors. It is a linear, high molecular weight glycouronan in

which three distinct types of segment are recognized (Painter, 1983; Kennedy & White, 1988).

These are blocks of β-(1→4)-D-mannuronic acid residues (MM block) and its epimer, α- (1→4)-

L-guluronic acid residues (GG block), and blocks which contain approximately equal quantities

of both types of uronic acid residue which may be randomly distributed or show some alternating

14

sequence. Other studies have explored the antitumor properties of red, brown and green algal

polysaccharides (Zhuang et al., 1995). For example, carrageenan, a family of sulfated galactan,

has shown to be active angiogenesis inhibitors.

Structure-activity relationships. Molecular weight, degree of branching, number of substituents,

water solubility and solution conformation significantly affect the biological activities of β-

glucans (Adachi et al., 2002). For example, the molecular weight and a triple-helical

conformation are known to be important factors for the immune-stimulating activity of lentinan.

The triple-helical lentinan with a moderate molecular weight (5.0x105~15.0x105) exhibits higher

anticancer activities against the growth of cancer cell such as sarcoma 180 than those with too

low or too high molecular weight (Zhang et al., 2005). In aqueous solution, β-glucans undergo

conformational change into triple helix, single helix or random coils. The immune functions of β-

glucans are apparently dependent on their conformational complexity (Bohn & BeMiller, 1995).

As for example, the triple-helical lentinan exhibits distinct antitumor activities but whenever the

triple helix has been broken into single random coils, its antitumor activity decreases

significantly or disappears. Therefore antitumor activity is related to triple helix conformation,

moderate molecular weight and its combined protein (Zhang et al., 2005; Unursaikhan et al.,

2006). However, the highly branched MD-fraction from G. frondosa (MW 1 000 000– 1 200 000

dalton) exerts a high antitumor activity (Nanba et al., 1987; Kodama et al., 2003). Higher

antitumor activity seems to be correlated with higher molecular weight, lower level of branching

and greater water solubility of β-glucans (Zjawiony, 2004).

Less is known about the structure-function relationship of acidic polysaccharides, although

the polyanionic properties of these macromolecules seem to be related to their antitumor activity.

Moreover, amongst alginates having very similar molecular weight (mol. wt. Range: 2.25-2.55 x

105) polymers with a higher content of MM-block showed higher antitumor activity than those

with a lower content despite they have the same poly-anionic properties (Fujihara & Nagumo,

1993). These facts imply that besides polyanionic properties some other features are responsible

for the biological activity of alginic acid. In living system the biospecific molecular recognition

is known to be based on the lock and key concept as proposed by Emil Fischer. On the basis of

this concept bioactive polysaccharides probably possess biospecific “keys” along polymer

backbone. Selected groups on the polysaccharide backbone represent keys, which are

macromolecular chains looped into potential cell surface receptors. Conformational flexibility of

15

Fig. 1.6. Block of β-(1→4)-linked L-mannuronic acid (M) residues.

OO

H

OH

HH

HOO

H

H

OHO

OO

H

OH

HH

HO

H

H

OHO

n

O

HOH

H

H OH

H

H

OO

HOH

H

H OH

H

H

O

OH

O

O

OH

O

n

the carbohydrate polymers facilitates an optimal adaptation to the extra cellular protein. The

molecular architecture of the D-mannuronan (Fig. 1.6) block is completely different from that of

L-guluronan block (Fig. 1.7), the former being twisted, whereas the latter being rigid and buckled

(Kennedy & White, 1988).

Now, in alginic acids of different origin the length of homoglycuronan segments are different

and therefore their three dimensional structure are also different. Alginates having higher

contents of MM-block have the right degree of conformational flexibility necessary for the

polymer-cell surface receptor interaction. Therefore, conformational factors related to the

distribution and sequence of the carboxyl groups on the alginate backbone may be responsible

for their variable biological activity.

Mechanism of action. The molecular mechanism of the inhibition and promotion processes in

carcinogenesis by plant polysaccharides is poorly understood. Polysaccharides or

polysaccharide-protein complex, having antitumor activity, can stimulate effector cells like

Fig. 1.7. Block of α- (1→4)-linked L-guluronic acid (G) residues.

16

macrophases, T-lymphocytes and natural killer (NK) cells to secrete cytokines like TNF-α, IFN-

γ, IL-1β, etc. These are antiproliferative and induce apoptosis and differentiation in tumor cell.

Research data showed that schizophyllan from S. commune can bind to mRNA poly (A) tail and

thus exert antitumor activity (Karinaga et al., 2004). In animal studies, after oral administration,

the specific backbone 1→3 linear β-glycosidic chain of β-glucans cannot be digested. Most β-

glucans enter the proximal small intestine and some are captured by the macrophages. They are

internalized and fragmented within the cells, then transported by the macrophages to the marrow

and endothelial reticular system. The small β-glucans fragments are eventually released by the

macrophages and taken up by other immune cells like the circulating granulocytes, monocytes

and dendritic cells via the complement receptor (CR)-3 leading to various immune responses

(Zhou & Gao, 2002). The immune response will then be turned on; one of the actions is the

phagocytosis of the monoclonal antibody tagged tumor cells. However, β-glucans of different

sizes and branching patterns may have significantly variable immune potency.

1-4.5. Antitussive activity

Recent studies showed that various naturally occurring carbohydrate polymers from medicinal

plants (Table 1.2) affect citric acid-induced cough reflex and reactivity of airways smooth

muscle in vivo conditions. For example, the polysaccharide materials from the leaves of popular

Malian medicinal plants Trichilia emetic (TE) and Opilia celtidifolia (OC), and fruits of

Crossopteryx febrifuga (CF) were able to suppress experimentally induced cough reflex in

guinea pigs. These crude polysaccharides are made up of arabinogalactan (~54%) and

rhamnogalacturonan (~30%) in T. emetic leaves, arabinogalactan (~60%), rhamnogalacturonan

(~14%) and glucuronoxylan (~14%) in O. celtidifolia leaves, and pectic polysaccharides (~75%)

together with arabinogalactan (~17%) in C. febrifuga fruits (Sutovska et al., 2009a). The fruits

extract of C. febrifuga was not active in the dose of 50 mg/kg b.w., however 10–20 fold higher

doses suppressed cough attack and also showed bronchodilatory properties. In addition, the

rhamnogalacturonan from the roots of Althaea officinalis L. showed potent biological effects on

the citric acid-induced cough reflex and reactivity of airways smooth muscle in vitro and in vivo

conditions. It possessed dose-dependent cough suppression effect comparable with opioid

agonist codeine (Sutovska et al., 2009b).

17

Class of carbohydrate polymer Source Dose

(mg/Kg)

System Reference

Arabinogalactan & rhamnogalacturonan Trichilia emetica 50 Guinea pigs Sutovska et al., (2009a)

Arabinogalactan, rhamnogalacturonan and Glucuronoxylan

Opilia celtidifolia 50 Guinea pigs Sutovska et al., (2009a)

Pectin &Arabinogalactan Crossopteryx

febrifuga

50 Guinea pigs Sutovska et al., (2009a)

Rhamnogalacturonan Althaea officinalis L. 50 Guinea pigs Sutovska et al., (2009b)

Pectin material with high arabinose and galacturonic acid

Opilia celtidifolia 50 Guinea pigs Sutovska et al., (2010)

Polysaccharide-polyphenolic conjugate Lythrum salicaria Sutovska et al., (2012)

Arabinogalactan Adhatoda vasica 50 Guinea pigs Chattopadhyay et al.,

(2011)

Arabinogalactan Withania somnifera 50 Guinea pigs Sinha et al., (2011b)

Arabinogalactan Glycyrrhiza glabra 50 Guinea pigs Saha et al., (2011)

Pectic polysaccharides pumpkin fruit biomass Guinea pigs Nosál’ová et al., (2011)

Extracellular proteoglycan Rhodella grisea Cats Nosál’ová et al., (2012)

Furthermore, water-soluble pectic polysaccharides rich in homogalacturonan and

rhamnogalacturonans branched with side chains containing arabinose and galactose residues

from Cucurbita pepo fruit, and leaves of Opilia celtidifolia biomass showed promising

antitussive activity.

Besides, arabinogalactan from several Indian medicinal plants including Adhatoda vasica

Table 1.2. Antitussive activity of carbohydrate polymers from natural sources

18

(Chattopadhyay et al., 2011), Withiania somnifera (Sinha et al., 2011b) and Glycyrrhiza glabra

(Saha et al., 2011) showed in vivo antitussive activities. This polysaccharide is consisted mainly

of (1,3)/-(1,6)/-(1,3,6) linked galactopyranosyl and (1,5)/-(1,3,5) linked arabinofuranosyl

residues.

Other natural macromolecules, which are not composed entirely of sugar units, contain blocks

of monosaccharide units as part of the molecular structure, and contribute extensively to their

antitussive activities. For example, a low molecular mass arabinogalactan-protein (AGP) from

the instant coffee powder of Coffea arabica beans, possess prominent antitussive (in vivo)

activity in a dose dependant way (Nosál’ová et al., 2011).

The mucilagineous extracellular proteoglycan (EPG) from culture medium of red alga

Rhodella grisea, which contained xylose and its 3-O-and 4-O-methyl-derivates (55%),

glucuronic acids (17%), rhamnose (14%), galactose (8%), glucose (4%) & contained protein

(13%) & minor amounts of other sugars (∼2%) also showed a cough suppressing effect on

laryngopharyngeal type of cough (Nosáľová et al., 2012).

The high molecular mass polysaccharide-polyphenolic conjugate (rhamnogalacturonan

associated with arabinogalactan in Lythrum conjugate) from flowering parts of Lythrum

salicaria that contains 74% of carbohydrates and 17% of phenolics showed antitussive activity

(Sutovska et al., 2012). The polyphenolic–polysaccharide–protein complex (molar mass

11.2 kDa) from flowers of Solidago canadensis L. composed of carbohydrates (43%), protein

(27%), phenolics (12%), uronic acids (10%) and inorganic material (8%) showed antitussive

activity (Sutovska et al., 2013).

Structure-activity relationships. The antitussive properties of polysaccharide from various

sources are different (Fig.1.8), but because of limited data available the relationship between the

structure of polysaccharides and their antitussive activity is not yet clear (Sutovska et al., 2010;

Nosáľová et al., 2011).

19

Mechanism of action. The mechanism behind the antitussive activity of the carbohydrate

polymers from plants is poorly understood, although it has been reported that many antitussive

herbs work by an antispasmodic action or bronchodilator (Ernst, 1998; Pavord & Chung, 2008).

They cause bronchial muscle relaxation in vitro, or decrease airways resistance in vivo. Pavord

(2004) reported that broncho constriction causes or enhances the sensitivity of cough, while

bronchodilation does the opposite. However, the role of other mechanism including bioadhesive

effect of the polysaccharide to the epithelial mucosa (Sutovska et al., 2009a) cannot be ruled out.

Further research should be directed in this area.

1-4.6. Antiviral activity

Many viruses display affinity for cell surface proteoglycans, especially heparan sulfate

proteoglycans, with high biological relevance to virus entry. This raises the possibility of the

application of sulfated polysaccharides in antiviral therapy. In recent years, screening assays of

the antiviral activity of extracts from a number of marine algae and cyano bacteria have led to

the identification of carbohydrate polymers with potent inhibitory effects against several human

and animal viruses, including Herpes simplex virus (Witvrouw & De Clercq, 1997; Balzarini &

Van Damme, 2007; Ghosh et al., 2009a; Rusnati et al., 2009).

0

1

2

3

4

5

6

7

8

9

Num

ber

of c

ough

effo

rts

(NE

)

Control

Codeine

Adhatoda vasica

Glycyrrhiza glabra

Withania somnifera

Rhodella grisea

Fig. 1.8. The influence of the carbohydrate polymer from Adhatoda vasica, Glycyrrhiza glabra, Withania somnifera, and Rhodella grisea,and codeine (positive control) and saline water (negative control) on the citric acid-induced cough efforts (NE) in guinea-pigs recorded at 30, 60, 120 and 300 min time intervals (Chattopadhyay et al., 2011; Saha et al., 2011; Sinha et al., 2011b; Nosál’ova et al., 2011).

N 30 60 120 300 Time interval (min)

20

Table 1.3. Carbohydrate polymers with antiviral activity

Families of

polysaccharide

Source Molecular weight Strain CC50

(µg/ml)

IC 50

( µg/ml)

Reference

Fucan Padina

tetrastromatica

50 kDa HSV-1 & HSV-2 1000 0.30-1.05 Karmakar et al.,

(2010)

Fucan Sargassum

tenerrimum

30 ± 5 & 26 ± 5 kDa HSV-1 1000 0.5-15 Sinha et al.,

(2010)

Xylogalactofucan

and Alginic acid

Sphacelaria

indica

26 ± 5 & 21 ± 5 kDa HSV-1 200 0.6-10 Bandyopadhyay

et al., (2011)

Xylogalactofucan

and Alginic acid

Laminaria

angustata

56 ± 5 & 32 ± 5 kDa HSV-1 1000 0.2-25 Saha et al.,

(2012)

Arabinogalactan Azadirachta

indica

80 kDa BoHV-1 >1600–1440 31.12 to

105.25

Saha et al.,

(2010)

Galactan Gracilaria

corticata

30 kDa HSV-1 & HSV-2 > 1000 1.1-27.4 Chattopadhyay et

al., (2008)

Xylan Scinaia hatei 120 kDa HSV-2 > 1000 0.22-1.37 Mandal et al.,

(2010)

Xylomannan Scinaia hatei 160 kDa HSV-1 > 1000 0.5-4.6 Mandal et al.,

(2008)

Xylomannan Sebdenia

polydactyla

150 kDa HSV-1 1000 0.35-2.8 Ghosh et al.,

(2009b)

Glucan Oryza sativa 1-30 kDa HCMV 270 3.46±0.63 Ghosh et al

(2010)

These polysaccharides include carrageenans, fucoidans, mannans, rhamnan sulfates, sulfated

galactans and others (Table 1.3). In addition, semisynthetic sulfated carbohydrate polymers from

dextran, cellulose and glucan also possess promosing antiviral activity.

Structure-activity relationships. Publications relating to antiviral activity of sulfated

polysaccharide demonstrate that the potency of these macromolecules depends upon the

following: (i) Degree of sulfation has a major impact on the antiviral activity of polysaccharides.

(ii) Specific position of sulfate groups might be important for antiviral activity. (iii) Molecular

weight contributes to antiviral activity. (iv) Antiviral potency also depends upon the molecular

21

structure of the polysaccharide. (v) Low-molecular weight compounds inhibit cell-to-cell spread

of viruses more efficiently than high molecular weight compounds (Ekblad et al., 2010).

Mechanism of action. The entry of HSV into host cells is a complex process initiated by the

specific interaction between host-cell-surface receptors and viral envelope glycoproteins

(Schneider-Schaulies, 2000; Spear, 2004; Kleymann, 2005; Olofsson & Bergstrom, 2005). In the

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 (Herold et al.,

1991, 1994; Cheshenko & Herold 2002). Clusters of basic and hydrophobic amino acids located

between residues 129 and 160 of gC1 (Trybala et al., 1994; Mardberg et al., 2001, 2002) as well

as the mucin-like region (amino acids 33–123) of this protein (Tal-Singer et al., 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 et al., 2006). Most of the variability between gB1 and gB2 is seen in a lysine-rich

region (amino acids 68–76), which is also responsible for binding to HS (Laquerre et al., 1998).

gB1 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 (Heldwein et al., 2006). The results from a more recent

study suggest that specific hydrophobic/aromatic amino acids from domain I are important for

the fusogenic activity of gB (Hannah et al., 2007). An attractive concept is that sulfated

polysaccharides act as antiviral agents in cell culture due to the fact that these charged polymers

may mimic HS chains on cell-surface proteoglycans and thus block viral attachment by

competitive inhibition.

A novel approach to inhibit HSV-1 infection by targeting the gD-mediated membrane fusion

step has been described (Copeland et al., 2008). The antiherpetic 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.

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. Finally, in

addition to the polysaccharide-mediated antiviral 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

22

intracellular events contributing to the antiviral 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 anticytomegalo viral effect of spirulan-like polysaccharides was

demonstrated to be composed of these two antiviral activities, i.e., an inhibition of HCMV entry

on the one side in addition to the induction of intracellular antiHCMV effects on the other side

(Rechter et al., 2006).The replication efficiency of most viruses is dependent on specific

intracellular signaling pathways, the inhibition or the induction of particular signaling by

surface-binding polysaccharides can provide a significant part of the overall antiviral activity.

One explanation for such intracellularly produced activity is the stimulatory effect of sulfated

polysaccharides onto interferon production with the consequence of a broad antiviral effect.

1-4.7. Potential clinical applications

During the past decades many significant developments in the utilization of carbohydrate

polymers as drug have been put forward. This, however, is not surprising since it is known that

many of these biopolymers play an essential role in key biological processes. Some of the new

cases of polysaccharide applications have been approved by the scientific world; others are still a

matter of controversies and hence are not being accepted for clinical approval (Witezak, 1995).

Many fungal and plant derived bioactive polysaccharides with a broad range of

immunomodulatory activities are found in traditional medicine. Some such polysaccharides have

been developed into drugs and show clinical efficacy in controlled trials while the majority of

such compounds remain as nutraceuticals with only preliminary research.

Three polysaccharide based carcinostatic (immunotherapeutic) agents, Krestin, Lentinan and

Sonifilan, have already been developed from mushroom (Borchers et al., 2004; Zhang et al.,

2011; Maehara et al., 2012). These are used currently in the treatment of cancer of the digestive

organs, lung and breast, as well as cancer of the stomach and cervical cancer, ovarian cancer

(Fujimoto et al., 2006), gastric or colorectal cancer, (Maehara et al., 2012) respectively. The

potential usefulness of bioactive polysaccharides in the treatment of diseases has been

demonstrated in preclinical and clinical studies on β-glucan. Such polysaccharides are generally

nontoxic and possess other bioactivities such as inducing differentiation, stimulating

hematopoiesis, antimetastasis, and antiangiogenesis, which make them ideal adjuvants in modern

23

cancer therapy. Again, other clinical experiments have showed the potential application of

lentinan on anti-HIV activity (Gordon et al., 1995, 1998).

For more than 70 years, heparin is a drug of choice in the prevention and treatement of

thromboembolic disorders (Linhardt et al., 1997; Gunay & Linhardt, 1999; Alban & Franz,

2001; Lee et al., 2008; Fan et al., 2011). In addition to heparin, partially depolymerized forms

called low molecular weight (LMW) heparins and a synthetic heparin pentasaccharide are

currently in clinical use (Murugesan et al., 2008). The subcutaneous injection of the LMW

heparins for the treatment of deep venous thrombosis has made anticoagulant therapy more

effective and easier to administer, making it possible to treat the patients without hospitalization

and at no increased risk of recurrent thromoembolism or bleeding complications (Rydberg et al.,

1999).

Although many polysaccharides and their functions have been described in this chapter, the

structure–function relationship remains to be elucidated. A great deal of fascinating chemistry

remains to be discovered. Elucidation of this interrelationship is the main objective of this work.

This research work has been presented into three parts. The first part provides an interesting

story about the structural features and antioxidant capacity of polysaccharides from the brown

seaweed Turbinaria conoides. The second part is devoted to structural features and antitussive

activity of water extracted polysaccharide from Adhatoda vasica. The final chapter describes the

materials and methods used in this study.

24

The goals of this research are to identify the polysaccharides present in Indian samples of

Turbinaria conoides and Adhatoda vasica and to study their biological activities.

The strategy adopted to achieve these goals involves:

� Isolation of the high molecular weight compounds.

� Their purification.

� Screening of the biological activities of the crude extracts as well as that of pure

compounds.

� Finally, determination of the structural features of the biologically active high

molecular weight compounds.

AIMS

RESULTS AND DISCUSSIONS

26

2

STRUCTURAL FEATURES AND ANTIOXIDATIVE

ACTIVITIES OF CARBOHYDRATE

POLYMERS FROM THE BROWN SEAWEED Turbinaria conoides

27

2-1. INTRODUCTION

Reactive oxygen species (ROS) are highly reactive molecules that are constantly produced by

enzymatic reactions in cells. In normal physiological conditions, ROS are produced at low levels,

which are necessary for maintaining normal cell functions and the endogenous antioxidative

defense systems of the body have the capacity to avert any harmful effects. However, free

radicals can escape from cellular defense system, leading to modification of DNA, proteins,

lipids and small cellular molecules are associated with a number of pathological processes,

including atherosclerosis, cancer and rheumatoid arthritis (Halliwell & Gutteridge, 1984).

Therefore, antioxidants are important for bodily protection against oxidative stress. Lipid

oxidation by reactive oxygen species (ROS) such as super oxide anion, hydroxyl radicals and

hydrogen peroxide also causes a decrease in nutritional value of lipids, in their safety and

appearance. In addition, it is the predominant cause of qualitative decay of foods, which leads to

rancidity, toxicity and destruction of biochemical components important in physiologic

metabolism. Recently, there is a considerable interest in the food industry and in the preventive

medicine for the development of antioxidants from natural sources, such as marine flora and

fauna, bacteria, fungi and higher plants. Among them, marine algae represent one of the richest

sources of bioactive compounds, and algae-derived products are increasingly used in medical

and biochemical research (Mayer & Lehmann, 2000). One particularly interesting feature of

marine algae is their richness in Sulfated polysaccharides, the uses of which span from food,

cosmetic and pharmaceutical industries to microbiology and biotechnology (Renn, 1997). These

macromolecules have been proven to show a wide range of biological activities important to

human health, for example, antiviral, antitumoral, antiinflammatory and anticoagulant activity

(Cumashi et al., 2007; Pomin & Mourao, 2008; Ghosh et al., 2009a).

In recent years, several classes of sulfated polysaccharides have been demonstrated to show

antioxidative activity, too. The compounds tested included glucan, alginic acid, fucoidan and

other unidentified macromolecules present in the extracts (Ruperez et al., 2002; Rocha de Souza

et al., 2007; Wang et al., 2008). Indian coastal area is inhabited by variety of marine algae which

are not yet to be fully explored and exploited for socio-economic development of the nation

(Wealth of India, 1985). Turbinaria conoides (Fucales, Sargassaceae) is one such seaweed,

which is widely distributed in the coastal areas of Andaman and Nicobar island, Laccadives,

Maharastra, Kerala, Tamilnadu, Gujrat, Andhra Pradesh (Sahoo, 2010). This brown alga contains

28

phytochemicals of great interest to researchers including highly cytotoxic hydroperoxysterol 24-

hydroperoxy-24-vinylcholesterol and fucosterol (Sheu & Sung, 1991), 24 ξ-hydroperoxy-24-

vinylcholesterol; 29-hydroperoxystigmasta-5,24(28)- dien-3β-ol; 24-ethylcholesta-4,24(28)-dien-

3-one; 24 ξ-hydroperoxy-24-ethyl cholesta-4, 28(29)-dien-3-one; 24-ethyl cholesta-4,24(28)-

dien-3,6-dione; 24 ξ-hydro peroxy-24-ethyl cholesta-4,28(29)-dien-3,6-dione; 6β-hydroxy-24-

ethyl cholesta-4,24(28)-dien-3-one; 24 ξ-hydroperoxy-6 β-hydroxy-24-ethylcholesta-4,28(29)-

dien-3-one (Sheu et al., 1999). The presence of two new antifungal steroids namely, 3,6,17-

trihydroxy-stigmasta-4,7,24(28)-triene and 14,15,18,20-diepoxy turbinarin have also been

reported (Kumar et al., 2010, 2011). Despite the general interest in phytoconstituents it remains

ironic that research on the chemical and biological aspects of the high molecular weight

bioactive has been neglected. Notably, it is intriguing to observe that polysaccharide from plant

extracts exhibit a large range of pharmacological activities (Inngjerdingen et al., 2006;

Mantovani et al., 2008; Ghosh et al., 2009a; Baek et al., 2010; Thakur et al., 2012).

Hence the central goal of this study was to investigate the structural features of the different

classes of polysaccharides present in T.conoides. We have also evaluated in vitro the

antioxidative activity of fucoidan, glucan and alginic acid isolated from this brown seaweed.

These polysaccharides may represent a new approach for inhibiting the harm caused by

excessive free radicals.

2-2. CHEMICAL CHARACTERIZATION OF POLYSACCHARIDES

FROM Turbinaria conoides

2-2.1. Preparation of depigmented algal power and sugar compositional analysis

The major goal of this study was to develop antioxidative drug candidate from Turbinaria

conoides. In order to study the chemical structures and antioxidative properties of polymers

present in T. conoides, the dried seaweed was sequentially extracted in a Soxhlet apparatus with

petroleum ether and acetone to leave a depigmented algal powder named as DAP (Fig. 2.1).

29

To understand the type of polysaccharides present in DAP, a knowledge about its sugar

composition is necessary. For the determination of the monosaccharide composition the fractions

obtained were hydrolyzed in 2M TFA, the sugars released were reduced and acetylated as

described in section 4-2.2. The alditol acetates were separated by GC and identified by their (i)

retention time relative to that of inositol hexa-acetate (present as internal standard) and (ii) mass

spectral fragmentation patterns. Fig. 2.2 shows a typical standard chromatogram and how mass

spectroscopy was used in order to verify the identities of alditol acetates.

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

min

m/z m/z m/z

Gal GlC Man Int. Std. (Inositol)

Xyl Fuc Rha

50 100 150 200 250 3000

25000

50000

75000

100000 43115 170129

9969 85 157145 187 23173 28920121741

303

50 75 100 125 150 175 200 2250e3

50e3

100e3

43

115

14510385

217127 18715873 20017555 218

50 100 150 200 250 300 3500

25000

50000

43

115

139103 187 217157170 25973 28941 361

5.0 7.5 10.0 12.5 15.0

0e3

500e3

1000e3

TIC Ara

Fig. 2.1. Scheme for the preparation of depigmented algal powder from the brown seaweed Turbinaria conoides.

Depigmented algal powder (DAP)

Sequential extraction with petroleum ether and acetone in Soxhlet apparatus

Air dried algal powder

Turbinaria conoides

30

GC analysis of the alditol acetates derived from DAP (Fig. 2.3) showed the presence of fucose

(56%), xylose (11%), galactose (15%), glucose (12%) and small amount of mannose (Table 2.1).

2-2.2. Isolation and sugar composition of polysaccharide fractions

Sugar composition of depigmented algal powder (DAP) revealed that, it contained 35% 2 M

TFA hydrolysable sugars of which about seventh were uronic acids. The main neutral sugar was

fucose and it also contained other sugar including glucose, galactose

5.0 7.5 10.0 12.5 15.0

0.0e6

5.0e6

10.0e6

15.0e6

20.0e6 T IC

Fig. 2.3. GC analysis of alditol acetates derived from DAP of Turbinaria conoides.

Fu

cose

Xyl

ose

Man

nose

Gal

acto

se

Glu

cose

Int.

Std

. (In

osi

tol)

min

SEC

Algal Powder (DAP)

0.1 M HCl, 30-350C

A

AF1

AF2

AF3 Residue

3% Aqueous Na2CO3 Extract B

2% CaCl2

Residue

INS

C

2% CaCl2

Precipitate (C2)

Solution (C1)

AEC

Water, pH~6.5, 800C

C1F1 C1F2

Fig. 2.4. Scheme for the extraction and purification of polysaccharides from the marine brown alga T. conoides.

31

and xylose (Table 2.1). The DAP was fractionated by the sequential extraction procedure as

shown in Fig. 2.4, was based on the different solubility, molecular mass and charge distribution

of polysaccharides from brown seaweeds. Glucans are soluble in warm waters and therefore

extracted at 80°C. Fucoidans were extracted with diluted hydrochloric acid, whereas alginates

were extracted with sodium carbonate. Alginates form insoluble precipitates with bivalent

calcium and at acidic pH, and are soluble in solution between pH 6 and 9. Procedures described

in the materials and methods (Section 4-1.1.).

GC and GC-MS analysis of the alditol acetates derived from A (Fig. 2.5) revealed the

presence of fucose as the major monosaccharide together with smaller amounts of xylose and

galactose (Table 2.1). No amino sugars were detected during GC-MS analysis of the derived

alditol acetates.

50 100 150 200 250 3000

25000

50000

75000

100000 43115 170129

9969 85 157145 187 23173 289201 21741

303

5.0 7.5 10.0 12.50.0e6

1.0e6

2.0e6

3.0e6

4.0e6 TIC

Gal

acto

se

Int.

Std

. (I

nos

itol)

Fu

cose

Xyl

ose

m/z

min

Fig. 2.5. GC-MS analysis of the alditol acetate derived from the fraction A of T. conoides.

32

Table 2.1. Yield and chemical composition (mol %) of fractions obtained from T. conoides

*percent weight of fraction dry weight.†mol percent of anhydro sugar - = not determined. nd = not detected. Tr = trace. TS = Total sugar

2-2.3. Purification of the fucoidans by chromatography

Anion exchange chromatography

on a DEAE Sepharose column

separated fraction A of T.

conoides into three sub-fractions

(AF1, AF2, and AF3) (Fig. 2.4).

AF1, which accounted for 11% of

the total sugars recovered from

the anion exchanger, was the

minor component of A. It

consisted mostly of fucose

together with smaller amount of

galactose, xylose and glucose (Table 2.1). The second fraction AF2, eluted at the beginning of

the salt gradient, is the second largest fraction. AF3 was the major fraction, amounting to 51% of

the total polymers recovered from the column. Fucose, accounted for more than 54% of the total

sugars of AF3 together with smaller amount of galactose and xylose units (Table 2.1). Thin layer

chromatographic analysis of the monosaccharides present in the hydrolysate indicates the

presence of an uronic acid with an Rf value

similar to that of glucuronic acid. GC analysis of

the TMS derivatives of the derived methyl

glycosides confirmed this result. The presence of

glucuronic acid has already been reported in

fucoidan from brown seaweeds (Adhikari et al.,

2006; Mandal et al., 2007).Therefore, AF3 is

essentially a fucoidan that might contain a high

number of sulfate groups, as indicated by its late

elution. Indeed, the high charge density of this

polysaccharide was confirmed by its sulfate

content (4%, w/w). The FT-IR spectrum

Fractions DAP A AF3 AF3D B C1 C1F2

Yield 100 8.8 nd nd 22.6 6.2 nd

TS* 35 36 37 56 46 41 58

Sulfate* nd 3 4 Tr 1 2 Tr

Protein* nd 1.3 nd nd 2.4 2.1 nd

Fucose† 56 57 54 52 Tr 59 6

Xylose† 11 15 18 19 Tr 12 5

Mannose† 7 Tr Tr - Tr Tr Tr

Galactose† 15 23 28 29 Tr 18 -

Glucose† 12 5 - - - 11 89

Fig. 2.6. FT-IR spectrum of fucoidan (AF3) obtained from T. conoides.

Sulfate band 1252 cm-1

33

0

0.5

1

1.5

2

2.5

3

00.0

80.1

50.23 0.3

0.380.45

0.53 0.6 0.68

0.75

0.83 0.9

0.98

Fig. 2.7. Elution profile of the fucoidan (AF3) of T. conoides on Sephacryl S-200 column with 500 mM sodium acetate buffer (pH 5.0) at 20 mL/h. Elution of polysaccharide 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].

Kav

Su

gar

Co

nce

ntr

atio

n (µg

/mL)

(Fig. 2.6) of AF3 showed an absorption band at 1252 cm-1 related to a >S=O stretching vibration

of the sulfate group. An additional sulfate absorption band at 848 cm-1 (C–O–S, secondary axial

sulfate) indicated that the sulfate group is located at position 4 of the fucopyranosyl residue

(Patankar et al., 1993).

Solvolytic desulfation of the purified (AF3) fucoidan (as pyridinium salt) produces a

desulfated derivative (AF3D). Preliminary experiments showed a higher recovery with this

method compared to methanol-HCl and auto-desulfation methods. In the IR spectrum of

desulfated derivative (AF3D) absorbances at 1252 cm−1 and 848 cm−1 become weak,

demonstrating that they were associated with sulfate groups. This purified sulfated fucan had

negative specific rotation [α]D -109o (c 0.2, H2O), revealing that fucose in AF3 belongs to the L-

series, like other sulfated fucans from brown seaweeds (Kariya et al., 2004; Adhikari et al.,

2006; Mandal et al., 2007). AF3 was subjected to further chemical analysis.

2-2.4. Molecular mass

AF3 was subjected to

further chemical analysis. First,

the apparent molecular mass

was determined by size

exclusion chromatography. The

elution profile of this

macromolecule on Sephacryl

S-200 suggests that this

polymer is homogeneous (Fig.

2.7). Based on calibration with

standard dextrans, the apparent

molecular mass of AF3 would

be 50 kDa.


Table 2.2 shows that the desulfated fucoidan yielded partially methylated alditol acetates

(PMAA) corresponding to (1→2)-Xylp (xylopyranose), (1→3)-linked Fucp, and (1→3)- and

(1→2,3)-linked Galp residues, consistent with the presence of fucoidan. The identity of each

34

PMAA was confirmed by their relative retention time and mass spectral fragmentation pattern.

Fig. 2.8 shows EI-MS fragmentation pattern of selected PMAA. The presence of large quantities

of T-Xylp (terminal xylopyranose) and T-Fucp residues suggest that this polymer is highly

branched with 29 terminals for every 71 residues in the main chain. Interestingly, 33–34% of the

total fucose residues are terminal, 27–28% are (1→3)-linked, 21–22% are branched points

(Table 2.2). Small amount of (1→4)- and (1→2)-linked Fucp residues were also present. So far,

fucose residues in algal fucoidans are either (1→3)- or (1→3)- and (1→4)-linked (Cumashi et

al., 2007; Pomin & Mourao, 2008). Linkage analysis of the native fucan sulfate AF3 yielded a

variety of monomethylated, dimethylated and trimethylated products (Table 2.2) indicating that

the structure of this polymer is highly complex. The amount of sulfate groups in the native

Methylation

products†

Deduced linkage Peak area* AF3 AF3D

2,3,4-Xyl Xylp(1→ 11 12

3,4-Xyl →2)Xylp(1→ 7 9

2,3,4-Fuc Fucp(1→ 8 17

2,4-Fuc →3)Fucp(1→ 3 14

2,3-Fuc →4)Fucp(1→ 3 3

3,4-Fuc →2)Fucp(1→ 5 6

2-Fuc →3,4)Fucp(1→ 17 5

3-Fuc

Fuc

→2,4)Fucp(1→

→2,3,4)Fucp(1→

2

16

6

-

3,4,6-Gal →2)Galp(1→ 6 7

2,4,6-Gal

4,6-Gal

2-Gal

→3)Galp(1→

→2,3)Galp(1→

→3,4,6)Galp(1→

10

6

6

10

6

5

polysaccharide as calculated from methylation was not in good agreement with the

experimentally determined sulfate. But, methylation of sulfated polysaccharides does not always

yield reliable proportions of methylated alditol acetates (Pereira et al., 1999; Adhikari et al.,

2006). 2-O-methyl fucitol, and unmethylated fucitol were amongst the abundant products of

methylation analysis of the native polymer. The increase in the proportions of 2,4-di-O-methyl

*Percentage of total area of the identified peaks. †2,3,4-Fuc denotes 1,5-di-O- acetyl-2,3,4- tri-O-methylfucitol, etc. - = not detected.

Table 2.2. Partially methylated alditol acetates derived from the fucoidan (AF3) of T. conoides and its desulfated derivative (AF3D)

35

fucitol after desulfation, as a consequence of decreased proportions of 2-O-methyl fucitol

residues, suggests that sulfate groups, when present, are located at position 4 of the (1→3)-linked

fucosyl residues. This result also confirms the prediction made by IR analysis regarding the

position of sulfate group.

CHDOAc

CHOMe

CHOAc

CHOMe

CHOAc

CH2OMe

118

161

129

234

102

CHOAc

CHDOAc

CHOMe

CH3

CHOMe

CHOMe

118

131 162 175 115

m/z 50 75 100 125 150 1750

5000

10000

15000

20000

25000

118

10289

13172

59

175162

CHDOAc

CHOMe

CHOMe

CHOMe

CH2OAc

118161

117162

102

101

(2)

m/z

Fig. 2.8. EI-MS fragmentation pattern of (1) 1,3,5-tri -O-acetyl-2,4-di-O-methyl fucitol; (2) 1,5-di-O-acetyl -2,3,4-tri-O-methyl fucitol; (3) 1,3,5 tri-O-acetyl-2,4,6-tri-O-methyl galactitol; (4) 1,5 di-O-acetyl -2,3,4 tri-O-methyl xylitol of T. conoides.

50 75 100 125 150 175 200 225 250 2750e3

50e3

100e3

150e3

43 118

129

101

16174 8759

234174143 203 217175 277

m/z

(1)

CHDOAc

CHOMe

CHOAc

CHOMe

CHOAc

CH3

247118

131234

174

m/z

(3)

50 75 100 125 1500

25000

50000

43101 117

59 8871 162131

(4)

m/z

36

HOD

H-6

H-1

α H

-1β

Chemical shift,

Fig. 2.9. 1H NMR spectrum at 500 MHz of the fucoidan (AF3) of T. conoides. The spectrum was recorded at 80°C for sample in D2O solution. H-1α, H-1β and H-6 refer to anomeric signals of α-linked fucose, β-linked sugars and methyl protons of fucose residues, respectively. The signal for the residual water was designated as HOD.

6

2-2.6. NMR spectroscopy

We employed 1H NMR spectroscopy to determine the anomeric configuration and the

sulfation pattern of the fucoidan AF3. The native

polysaccharide has a very complex 1H NMR

spectrum (Fig. 2.9). A number of separate spin

systems ranging from 5.02 to 5.43 ppm

attributable to anomeric protons of α-fucose

residues were distinguishable in the spectrum of

the pure fucoidan (Mourao et al., 1996; Kariya et

al., 2004; Mandal et al., 2007). The appearance

of large number of anomeric signals suggest that

the environment of the fucose residues are

different and this may arise from varied

sulfation pattern, different glycosidic

linkage positions and/or diverse sequence

of monosaccharide residues. This spectrum

also included resonances characteristic of

fucoidan, such as signals from ring protons (H-2 to H-5) between 3.34 and 4.34 ppm, and intense

signals from the methyl protons H-6 at ~1.02–1.28 ppm. This later envelope of signals confirms

the presence of different types of fucosyl residues. The signals between 4.45 and 4.90 ppm were

attributed to anomeric protons of β-linked sugars. The high proportion of xylose and galactose

residues must be responsible for these signals in the spectrum, but was not possible to assign any

particular signals to these residues. Therefore, it is apparent that the NMR spectrum of this

polysaccharide is complex as observed for fucoidan from other marine brown algae (Mourao et

al., 1996; Pereira et al., 1999; Adhikari et al., 2006; Mandal et al., 2007).

ppm

37

p

G M G

HOD

Chemical shift, ppm Fig. 2.11. 1H NMR spectrum at 500 MHz of the alginic acid containing fraction (B)

isolated from the brown seaweed T. conoides. G1 and M1 are the anomeric signals of

guluronic acid and mannuronic acid, respectively, and G5 is the signals of the H-5 proton of

guluronic acid residue. The spectrum was recorded at 80°C for sample in D2O solution. The

signal for the residual water was designated as HOD.

HOD

0

0.2

0.4

0.6

0.8

1

1.2

0.00

0.00

0.07

0.13

0.20

0.26

0.33

0.40

0.46

0.53

0.59

0.66

0.73

0.79

0.86

0.92

0.99

Sug

ar

Co

nce

ntra

tion

(µg/m

L)

Kav.

Fig. 2.10. Elution profile of the hot water extracted fraction (C1) of T. conoides on Superdex-30 column with 500 mM sodium acetate buffer (pH 5.0) at 20 mL/h. Elution of polysaccharide 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].

2-2.7. Structural features of the glucan and alginic acid rich fractions

Glucans are soluble, but their water

solubility depends on the branching level: the

higher the branching degree, the higher the

solubility. The less branched glucans are

soluble only in warm water (60–80°C). The

alginic acid (C2) present in the hot water

extract (C) was removed by taking advantage

of the insolubility of Ca- alginate in water.

The molecular mass of glucans is small (5–10

kDa). Therefore, attempts have been made to

separate it from fucoidan by size exclusion

chromatography. Indeed, SEC

on Superdex-30 separates C1 into two fractions C1F1 and C1F2 (Fig. 2.10). The minor fraction,

C1F2, which accounted for 35% of the

total sugar recovered from the column

consisted of mainly glucose as neutral

sugar. Based on calibration with

standard dextrans, the apparent

molecular weight of this glucan would

be 5 kDa. This glucan containing

fraction (C1F2) does not produce blue

colouration with iodine. Moreover 1H

NMR spectrum of this polymer clearly

shows the presence of only one

anomeric signal at 4.827 ppm indicative

of β-configuration. Finally, methylation

analysis of this glucan revealed

the presence of 1,3,5-tri-O-

acetyl-2,4,6-tri-O-methylglucitol

C1F1

C1F2

ppm

38

(98%) and 1,5-di-O-acetyl-2,3,4,6-tera-O-methyl glucitol (2%) residues. On the basis of the

glycosidic linkage and 1H NMR analysis it may be concluded that the glucan of T. conoides is a

linear polysaccharide and contained β-(1→3)-linked glucopyranosyl residues. Sodium alginates

form insoluble precipitates at acid pH and with calcium salts, but they are soluble in solution

between pH 6 and 9. Therefore, these macromolecules were extracted with Na2CO3. The

fractional product (B) was analysed using FT-IR and 1H NMR spectroscopy. The FT-IR

spectrum of this fraction contains band at 3400 (OH stretching), 2925 (CH stretching), 1675 and

1420 (COO stretching) cm-1 characteristic of alginate. Moreover two bands at approximately

1100 and 1025 cm-1 responsible for mannuronic (M) and guluronic (G) units, respectively, were

also observed. To evaluate the content of G, M and G–G linkage, we have investigated the 1H

NMR spectrum (Fig. 2.11) of sodium alginate using procedures as described by Grasdalen, 1983

and Matsushima, 2005. The relative areas of anomeric protons G-1 (H-1 of G) and M-1 (H-1 of

M) correspond to the mole fractions of G and M, respectively. The peak areas of G-5 (H-5

resonances of G) give the distribution of G in G-block and GM-block, and the algebraic sum of

their intensities accounts for the total G content, i.e., the relative area of G-5 is equal to that of G-

1. From the measurement of peak areas of A (δH 5.13 (G-1)), B (δH 4.72–4.84 (G-5 in GM-

block and M-1)) and C (δH 4.45–4.52 (G-5 in G-block)), the guluronic acid content (G%) and

G–G diad frequency (GG%) were calculated using Eqs. (1) and (2), and the calculated values are

46.19 and 42.12, respectively

2-3. PHARMACOLOGICAL ACTIVITIES OF POLYSACCHARIDES

FROM Turbinaria conoides


Ferric ion reducing/antioxidant power (FRAP) assay. In recent years many different methods

are being used to evaluate antioxidative capacity of foods and biological samples (Huang et al.,

(2) (area of B) + (area of C)

GG% (%) = area of C X 100

(1) (area of B) + (area of C)

G% (%) = area of A X 100

39

Table 2.3. Ferric ion reducing abilitya of polysaccharide containing fractions from T. conoides

4 min 30 min

Fractions µµµµmol

Trolox/g

µµµµmol

Fe(II)/g

µµµµmol

Fe(II)/g

µµµµmol

Trolox/g

AF3b 165 79 190 96

Bb 92 53 137 85

C1F2 b 72 38 82 43

aResults are expressed as equivalent of Fe(II) or Trolox per gram dry weight of fractions in aqueous solution. Each value, which is given to its nearest whole number, was the mean of 2 replicates. bFor a description of fractions obtained see “Materials and methods”.

2005). In some of these protocols, antioxidative assays were performed in alcoholic solutions,

but in this condition polysaccharide

would precipitate. Therefore,

the antioxidative capacity of

sulfated polysaccharides (AF3, B

and C1F2) from Turbinaria

was determined by the FRAP

assay. This assay in which a ferric

salt Fe(III) (TPTZ)2Cl3

(TPTZ=2,4,6-tripyridyl-s-triazine)

is used as oxidant (Benzie &

Strain, 1996) takes advantage of

electron transfer reactions (Huang et al., 2005). FRAP values increased considerably from 4 to

30 min, as it has been described for other vegetable and seaweed samples (Ruperez et al., 2002).

Regarding antioxidative capacity of the polysaccharides of present study it is clear that the

fucoidan (AF3) showed the highest reducing power at 4 and 30 min (165 and 190 mol Fe(II) per

g sample dry weight, respectively). This is followed by alginic acid and glucan (Table 2.3).

Results are expressed as mol Fe(II) per g sample dry weight. For comparison of potencies, values

are also calculated as mol Trolox/g sample dry weight from regression equations as described by

Pulido, 2000 of Trolox at 4 and 30 min of reaction with the FRAP reagent.

Scavenging effect on1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. For further insight into

the activation mechanism, we examined whether the protective effect was associated with DPPH

radicals. The proton radical scavenging action is known to be one of the various mechanisms

40

Fig. 2.12. Scavenging effect of polysaccharides isolated from the brown seaweed T. conoides on DPPH radicals. BHA: Butylated hydroxyanisole. AF3: The fucoidan rich fraction. B: The alginic acid containing fraction. C1F2: The glucan fraction.

0 1 2 3 4 5

0

20

40

60

80

100

BHA AF3 B C2F3

Sca

veng

ing

effe

cts

(%)

Concentration (mg/ml)

C1F2

for antioxidation. DPPH is one of the compounds that possess a proton free radical and shows a

characteristic absorption at 517 nm (purple) (Matsukawa et al., 1997). When DPPH encounters

proton radical scavengers, its purple colour would fade rapidly (Yamaguchi et al., 1998). An

excellent scavenging capability on DPPH radicals at a dosage of 1 mg/ml (61%) was found with

the fucoidan fraction AF3 as compared with the control BHA (100%) regarding the low dosage

ranges used (Fig. 2.12). More significant and effective radical scavenging capability (90%) was

also found with the fraction AF3 at a

higher dosage (5 mg/ml).

Comparable results with the fraction

AF3 were found to be 62% at the

dosages of 5.0 mg/ml for the alginate

rich fraction B (Fig. 2.12). In

contrast, the glucan fraction C1F2

demonstrated least scavenging

capability (50%) among three

fractions at the concentration of 5.0

mg/ml. Moreover, the marked

inhibitory effect of these

polysaccharides on DPPH radicals was

found to be concentration dependent (Fig. 2.12), though the activities were low. These results

reveal that the polysaccharides of present study are potent scavenger and their antioxidative

activity may be attributed to their proton-donating ability (Shimada et al., 1992). Probably, these

fractions might contain a higher amount of reductone which could react with radicals to stabilise

and terminate radical chain reaction. Whether the structures can be related to scavenging

capacity for DPPH radicals remains to be determined in future studies. However, our preliminary

results suggest that different bioactivities of these fractions apparently may be, in some respects,

linked to their different molecular structures. Most of earlier data on antioxidants of foods and

biological samples were on phenolic compounds and many researchers have been reported

positive correlation between free radical scavenging activity and total phenolic compound. But

this correlation between structure and activity is not always valid. For example, selected

enzymatic extracts of seaweeds (Ecklonia cava and Sargassum coreanum) did not possess

41

antioxidative activity, although they contained as much phenolic compounds as the other extracts

of E. cava. Feruloyl oligosaccharides showed a higher antioxidative capacity than free ferulic

acid (Yuan et al., 2005). Moreover, the former showed greater antioxidative capacity in vivo than

in vitro when compared with vitamin C (Ou et al., 2007). Therefore, it is believed that other

materials in seaweed extracts, such as small molecular weight polysaccharides, pigments,

proteins or peptides, may influence the activity. Indeed, recent data showed that a number of

polysaccharide containing fractions isolated from various sources such as medicinal mushrooms

(Shimada et al., 1992; Jiang et al., 2008), higher plants (Aguirre et al., 2009), marine algae

(Ruperez et al., 2002; Rocha de Souza, 2007; Wang et al., 2008, 2009a) and even some

enzymatic extracts (Je et al., 2009) possess antioxidative activity. Our results are in agreement

with these studies. In addition, the polysaccharides used in this study were purified and,

therefore, conclusively prove their antioxidative potency. Literature data also shows that some

polysaccharides have higher antioxidative potency than the others. For example, the

antioxidative potential of sulfated polysaccharides from the brown seaweed Fucus vesiculosus

was higher than that of agar-like sulfated galactans from the red seaweed Nori (Ruperez, 2001).

In a more recent study this group (Ruperez et al., 2002) found that antioxidative potency of

fucoidan containing fraction from the brown alga Fucus vesiculosus was higher than that of

glucan and alginic acid containing fractions. The glucan (C1F2) and alginate (B) rich fractions of

present study exhibited lower antioxidative potentials than the fucoidan containing fraction AF3

(Table 2.3). This result is in agreement with that of (Ruperez et al., 2002) except that the

antioxidative potency of the purified polysaccharides of present study is lower. Although the

antioxidative capacity of polysaccharide has been proven, the relationships between structure and

antioxidative capacity have not yet been elucidated. This is primarily due to the interplay of two

important factors. Firstly, the huge structural diversity of these polysaccharides has given a

major hindrance in the structure–activity relationship establishment. However, on the basis of the

accumulated data several common structural motifs emerge that are important for activity.

Secondly, many of the hitherto used sulfated polysaccharides contained a number of other

molecules. These later molecules may have their own activities, and or at least dilute the efficacy

of the sulfated polymer itself. However, the polysaccharides described in this work are purified

and therefore, conclusively proves their antioxidative activities.

42

2-4. CONCLUSIONS

In this study, we found that the soluble polysaccharides of T. conoides possess antioxidative

property. The higher molecular mass fucoidan (50 kDa) exhibits stronger activity than the low

molecular mass glucan (5 kDa) and, therefore, this potency probably be directly related with the

molecular mass of these polysaccharides. These differences can as well be attributed to the

differences in structure. The antioxidative activity of these polysaccharides may be attributed to

their proton-donating activity as evidenced through DPPH radical scavenging results. Because

the polysaccharides tested in this study was basically prepared without toxic chemical reagents, it

can be assumed to be potentially useful as a safe antioxidants for food processing industries.

Furthermore, as the isolation of these polysaccharides involves a few inexpensive and easy steps

it will be of an added advantage.

3

STRUCTURAL FEATURES AND ANTITUSSIVE

ACTIVITIES OF A CARBOHYDRATE POLYMER FROM

Adhatoda vasica

44

3-1. INTRODUCTION

Cough is a protective reflex that is vital to remove foreign material and secretions from the

airways (Nasra & Belvisi, 2009). In the physiological conditions in healthy persons this reflex

serves its function appropriately. However, modulation of the cough reflex pathway can lead to

inappropriate coughing and an augmented cough response. Ineffective cough is associated with

respiratory morbidity such as recurrent pneumonia. However, chronic cough can be troublesome.

It impairs the quality of life in adults (French et al., 2002) and significantly worries the parents

of coughing children (Cornford et al., 1993). Therefore, cough is the most common condition for

which patients seek consultation from a doctor (Schappert & Burt, 2006). Codeine and

dextromethorphan are extensively used for the treatment of cough (Chung, 2009). But current

antitussives, such as opiods, have unwanted side-effects (Belvisi & Hele, 2009). Therefore, new

antitussive agents are needed. In this context, natural products scaffolds have been invaluable as

biologically validated platforms for drug development (Ramallo et al., 2011).

Adhatoda vasica, also known as Basak is a shrub belonging to the Acanthaceae family. This

small evergreen shrub has been used in traditional Indian medicine for more than 2000 years

(Wealth of India, 1988; Kapoor, 1990). Leaves of this shrub are the main source of drug

preparation. The juice from the leaves and a decoction or infusion of the leaves and roots, water

extracts or syrups are used as herbal remedy for asthma, bronchitis, and chronic coughs and

breathlessness (Amin & Mehta, 1959; Dhuley, 1999). Basak contains alkaloids of great interest

to researchers including vasicine, vasicinone, deoxyvasicine, vasicol, adhatodinine, vasicinol and

others (Claeson et al., 2000). Other constituents include the vitamins, saponins, flavonoids as

well as steroids, and fatty acids (Wealth of India, 1988). As for example, roots contain a steroid,

called daucosterol, carbohydrates and alkanes (Huq et al., 1967; Jain et al., 1980; Jain & Sharma,

1982). Flowers were shown to harbor a triterpene α-amyrin; flavonoids (astragalin, kaempferol,

quercetin, etc) and alkanes (Huq et al., 1967; Claeson et al., 2000). Two aliphatic

hydroxyketones were also isolated from the aerial parts of A. vasica, namely, 37-

hydroxyhexatetracont-1-en-15-one and 37-hydroxyhentetracontan-19-one (Singh et al., 1991).

Two new pyrroloquinazoline alkaloids [1,2,3,9-tetrahydropyrrolo(2,1-b-quinazolin-9-one-3R-

hydroxy-3(2'-dimethylamino phenyl (desmethoxyaniflorine) and 7-methoxy-3R-hydroxy-1,2,3,9-

tetrahydropyrrolo[2,1-b]-quinazolin-9-one (7-methoxyvasicinone)] were also isolated from A.

45

vasica and their structures were deduced using spectroscopic and X-ray diffraction analyses

(Thappa et al., 1996). Four quinazoline alkaloids anisotine, 3-hydroxy-anisotine, vasicolinone,

vasicoline and one isoquinoline alkaloid 1-phenyl-2-methyl-6,7-dimethoxy-1,2,3,4-

tetrahydroisoquinoline were also isolated from the leaves of A. vasica. Additionally

unsaponifiable fraction of petrol ether extract of leaves were shown to contain higher levels of

beta-sitosterol and tricontane, whereas saponifiable fraction contains higher levels of linolenic,

arachedonic, linoleic, palmitic and oleic acids (Abd El-Megeed Hashem & Ahmed Elsawi,

1998). A new triterpenoid, 3 α-hydroxyoleanane-5-ene was also isolated from the aerial part of

A. vasica (Sultana et al., 2005). Some of these compounds contribute to the observed medicinal

effect of this plant. For example, the alkaloid vasicine showed bronchodilatory activity both in

vitro and in vivo. Vasicinone showed bronchodilatory activity in vitro but bronchoconstrictory

activity in vivo (Atal, 1980). Notably, these compounds cannot be used during pregnancy.

However, there have been no reports on the high molecular weight bioactive compounds present

in A. vasica although plant extracts consisting of carbohydrate polymers exhibit a large range of

pharmacological activities (Inngjerdingen et al., 2006; Cumashi et al., 2007; Sutovska et al.,

2007, 2009; Mantovani et al., 2008; Ghosh et al., 2009a; Baek et al., 2010; Thakur et al., 2012).

The present study reports isolation and chemical characterization of a water extracted

polysaccharide isolated from A. vasica leaf. Using chemical and chromatographic methods we

have been able to deduce structural features of a pectic arabinogalactan. We have also

investigated the antitussive activity of this polysaccharide in terms of number of cough efforts on

the citric acid-induced cough reflex and the reactivity of the airway smooth muscle in vivo

conditions in guinea pigs.

46

Table 3.1. Sugar composition and protein content of the water extracted polysaccharides containing fraction (WE) isolated from A. vasica leaves, the arabinogalactan protein (AGP) and of fractions (F1 – F3) derived by size exclusion chromatography

WE AGP F1 F2 F3 Yielda - - 16 72 12 Total sugarb 49 ndd 51 49 47

Uronicacidb 7.3 ndd 7.6 7.2 6.9

Proteinb Rhamnosec

21 2

ndd 1

19 3

21 2

22 2

Arabinosec 29 32 29 30 30

Xylosec 1 tre 1 1 tre

Mannosec 1 1 1 tre tre

Galactosec 60 66 60 62 61

Glucosec 7 tre 6 5 7

apercent weight of total neutral sugar recovered. bpercent weight of fraction dry weight. cmol percent of neutral sugars. ndd, not determined. tre, trace.

3-2.CHEMICAL CHARACTERIZATION OF THE PECTIC ARABINOGALACTAN FROM Adhatoda vasica

3-2.1. Isolation and chemical composition

The objectives of this research were to analyze the water extracted polysaccharide generated

from the medicinal plant Adhatoda vasica and to study its antitussive activity. In Indian

Ayurvedic system of medicine a decoction of its leaves in water is used as herbal remedy for

chronic coughs and breathlessness (Amin, 1959), therefore fresh leaves of this evergreen shrub

were extracted with water.

The yield of the water extracted polymer (named as WE), after fractional precipitation with

ethanol, was 10mg per gram of fresh leaves (Fig. 3.1). The use of cold water, in principle, may

exclude the extraction of physiologically inactive starch, present in the leaves. Fraction WE

contains 49% (w/w) neutral sugar along with 21% (w/w) protein. The amino acid composition of

protein associated with

fraction WE showed that

glutamic acid / glutamine

(38.5%), alanine (13.3%),

serine (9.4%), and glycine

(6.5%) were the major

constituents. The uronide

content of this fraction is

7.3% (w/w). Thin layer

chromatographic analysis of

the monosaccharides present

in the hydrolysate indicates

Fig. 3.1. Scheme for the isolation and purification of carbohydrate polymers from Adhatoda vasica.

Water, (25-32)°C WE

Size Exclusion Chromatography

Sephacryl S-100

F1

F2

F3

47

Fig. 3.2. Elution profile of the water extracted polysaccharide obtained from Adhatoda vasica leaves on Sephacryl S-100 column with 500 mM sodium acetate buffer (pH 5.5) at 30 ml/h. Collected fractions were analyzed for total sugar content by phenol-sulfuric acid. Elution of polysaccharide was expressed as a function of the partition coefficient Kav [Kav = (Ve-Vo)/(Vt-Vo) with Vt and Vo being the total and void volume of the column determined as the elution volume of potassium hydrogen phthalate and dextran (500 kDa), respectively and Ve is the elution volume of the sample].

F2

F1

F3

the presence of an uronic acid with Rf values similar to that of galacturonic acid. GC analysis of

the TMS derivatives of the derived methyl glycosides confirmed this result, but it also shows the

presence of traces of glucuronic acid. Sugar compositional analysis revealed that fraction WE

consist mainly of arabinose and galactose as the major neutral sugar together with smaller

amount of glucose, rhamnose, mannose and xylose units (Table 3.1). Considering that the water

extracted polymeric fraction (WE) contained galactosyl and arabinosyl residues as the major

sugars, and its protein content is 21%, we have tested its reactivity with β-glucosyl Yariv reagent

that specifically precipitates AGPs. We found that the major part of WE was Yariv soluble.

Sugar compositional analysis of this precipitate (named AGP) shows that it consisted mainly of

galactose residues and, to a lesser extent, arabinose residues, confirming the presence of AGP

(Table 3.1). This material also contained mannose residues probably originating from N-glycans.

3-2.2. Size exclusion chromatography (SEC)

SEC of WE on Sephacryl S-100 yielded three

overlapping subfractions (F1, F2 and F3; Fig.3.2). The

yield and chemical composition of these subfractions

are given in Table 3.1. They had similar

monosaccharide compositions. The only difference

between these three samples, as judged by size

exclusion chromatography, seems to be the molecular

weight. Based on calibration with standard dextrans,

the apparent average molecular mass of

macromolecules present in subfractions F1, F2

and F3 would be 61, 42 and 9 kDa, respectively.

48

Table 3.2. Partially methylated alditol acetates derived from the water extracted polysaccharide fraction (WE) of A. vasica leaves

Methylation products a

Sugar Linkagesb

m/z values Peak areac

2,3,5-Ara Terminal-Araf 43,45,102,118,129,161,205 trd

2,3-Ara 1,5-Arap 43,102,118,129,162,189,233 10

2-Ara 1,3,5-Arap 43,118,201,261 12

2,3-Xyl 1,4-Xylp 43,87,102,118,129,162,189,233 1

3,4-Rha 1,2-Rhap 43, 130,131,174,175,190,234 1

3-Rha 1,2,4-Rhap 43,130,143,190,203 1

2,3,6-Glc 1,4-Glcp 43,45,102,113,118,129,130,162,233 13

2,3,4,6-Gal Terminal-Galp 43,45,101,102,117,118,129,145,161,162, 205 4

2,4,6-Gal 1,3-Galp 43,45,101,118, 129,161,174,234 17

2,3,6-Gal 1,4-Galp 43,45,102,113,118,129,130,162,233 4

2,3,4-Gal 1,6-Galp 43,102,118,129,162,189,233 5

2,4-Gal 1,3,6-Galp 43,118,174,189,234 32

a 2,3,5-Ara denotes 1,4-di-O-acetyl-2,3,5-tri-O-methylarabinitol, etc. b Terminal-Araf denotes terminal arabinofuranosyl unit, etc. c Percentage of total area

of the identified peaks. trd = trace.


Methylation analysis of the polysaccharide from A. vasica yielded a variety of partially

methylated alditol acetates (Table 3.2). The results suggest that galactopyranosyl residues are

1,3- and 1,3,6-linked, whereas arabinofuranosyl units are 1,5- and 1,3,5- linked. Fig. 3.3 shows

EI-MS fragmentation pattern of selected molecules. The presence of 1,2- and 1,2,4-linked

rhamnopyranosyl residues was also indicated. This result suggests the presence of pectic

arabinogalactan. This fraction also contained 1,4-linked glucopyranosyl residues probably

originating from starch.

49

50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.00

10

20

30

%43 118

129

87101 18959

12874 97 139 234160 17456 202185 219

CHDOAc

CHOMe

CHOAc

CHOMe

CHOAc

CH2OMe

118

161

129

234

50 75 100 125 150 175 200 225 250 2750e3

50e3

100e3

150e3

43 118

129

101

16174 8759

234174143 203 217175 277

m/z

m/z

CHDOAc

CHOMe

CHOAc

CHOMe

CHOAc

CH2OAc

118

189

129

234

50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.00.0

25.0

50.0

%43

118

12987102

149 1899959 7146 162 223143 203173 233 249

m/z

50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0 300.00.0

25.0

50.0

%43

118

44 998559 127 261201160 173 190141 217 235 245 309

m/z

CHDOAc

CHOMe

CHOMe

CHOAc

CH2OAc

118

189162

129

CHDOAc

CHOMe

CHOAc

CHOAc

CH2OAc

118

261

Fig. 3.3. EI-MS fragmentation pattern of (1) 1,3,5 tri- O-acetyl -2,4,6-tri-O-methyl galactitol; (2) 1,3,5,6-tetra-O-acetyl -2,4-di-O-methyl- galactitol; (3) 1,4,5- tri-O-acetyl-2,3-di-O-methyl arabinitol; (4) 1,3,4,5 tetra-O-acetyl-2-O-methyl arabinitol of A. vasica.

(1)

(2)

(3)

(4)

50

3-3. ANTITUSSIVE ACTIVITY OF THE PECTIC ARABINOGALA CTAN FROM Adhatoda vasica

3-3.1. Assessment on chemically induced cough and airways defense reflexes

The effect of water extracted pectic arabinogalactan from A. vasica (WE) was evaluated on

the citric acid-induced cough reflex and reactivity of airways smooth muscle in vivo conditions.

For comparative purposes, codeine was simultaneously assayed as known reference compound.

The results of antitussive test showed that peroral administration of the pectic arabinogalactan in

a dose 50 mg kg−1 body weight brought about a significant decrease in the number of citric acid

induced cough efforts (NE) in adult healthy awaken guinea-pigs (Fig. 3.4). The first statistically

significant result was observed within 30 min after application. Furthermore, this positive effect

was observed during all the study time intervals. Notably, the suppression of cough efforts by the

polymer was quantitatively similar to that of codeine. Airway resistance is a concept used in

respiratory physiology to describe mechanical factors which limit the access of inspired air to the

pulmonary alveoli, and thus determine airflow. It is dictated by inter alia, the diameter of the

airways.

At present the relationship between cough and bronchoconstriction is not known with

**

**

** **

0

1

2

3

4

5

6

7

8

9

N 30 60 120 300 min

Nu

mb

er o

f co

ug

h e

ffo

rts

(NE

) control

codeine

WE

**

***

**

***

**

***

**

***

Fig. 3.4.The influence of the pectic arabinogalactan from Adhatoda vasica (WE), codeine (positive control) and saline water (negative control) on the citric acid-induced cough efforts (NE) in guinea-pigs recorded at 30, 60, 120 and 300 min time intervals. All used substances were applied by peroral route of administration: plant polysaccharides in the dose of 50 mg kg-1, codeine in the dose 10 mg kg-1and saline water in the dose 1 ml kg-1body weight. N, Initial values before application of the polysaccharides and codeine. Statistical significance

51

N 30 60 120 300 min0

5

10

15

20

25

R x

V (cm

H2O

s-1) Contr

Codeine

WE

certainty. Although it is generally accepted that bronchodilating substances can cause cough

suppression. Therefore, we have evaluated the changes of specific airway resistance as indictor

of this activity. Our result suggests that the application of arabinogalactan from A. vasica in the

dose which provoked cough suppressive activities did not significantly change the values of

specific airway resistance (Fig. 3.5). It also showed that after 30 min from application of

polysaccharide sample a slightly decreased value of specific airway resistance is registered.

Notably, in this time interval application of codeine provoked the same result. Recent studies

showed that several naturally occurring polysaccharides possess antitussive activity (Nosál’ova

et al., 2005; Sutovska et al., 2007, 2009a; Saha et al., 2011; Sinha et al., 2011b). The most potent

antitussive activity is observed with polysaccharide containing the highest proportion of uronic

acid constituent (Sutovska et al., 2007), although neutral polysaccharides may also have

significant activity (Nosál’ova et al., 2005). Therefore, the fact that the pectic arabinogalactan

from A. vasica modulates chemically induced cough reflex and specific airway resistance is not

surprising.

3-3.2. Mechanism of action

The mechanism behind the antitussive activity of the pectic arabinogalactan of present study

is not known, but many antitussive herbs probably work through an antispasmodic action or as

bronchodilator (Ernst, 1998; Pavord & Chung, 2008). They cause bronchial muscle relaxation in

Fig. 3.5. The influence of water extracted polysaccharide from the medicinal plant Adhatoda vasica and control agents (vehicle and codeine) on citric acid induced changes of specific airway resistance (R x V) in vivo conditions, registered before any agent application (values labelled as N in graphs) and after that in 30, 60, 120 and 300 min time intervals.

52

vitro, or decrease airways resistance in vivo. The arabinogalactan of present study possesses very

high cough suppressive effect and decreases the values of specific airway resistance in vivo

conditions only slightly. Pavord (2004) reported that bronchoconstriction causes or enhances the

sensitivity of cough, while bronchodilation does the opposite. However, the role of other

mechanism including bioadhesive effect of the polysaccharide to the epithelial mucosa (Sutovska

et al., 2009a) cannot be ruled out.

3-4. CONCLUSIONS

This study represents the first account on the in vivo antitussive activity of the water extracted

polysaccharide and its structural features. Biological investigations indicated that the pectic

arabinogalactan of A. vasica displayed promising activity in the antitussive assays. Moreover, as

the macromolecule tested in this study was basically isolated without toxic chemical reagents, it

can be assumed to be potentially useful as a safe antitussive agent for industries. Furthermore, as

the isolation of these polysaccharides involves few inexpensive and easy steps it will be of an

added advantage. Finally, the biological activity observed in A. vasica provides a scientific basis

for the use of the plant in traditional medicines.

EXPERIMENTAL SECTION

54

4

MATERIALS AND

METHODS

55

4-1. PLANT MATERIALS AND EXTRACTION OF POLYSACCHARI DES

4-1.1.Isolation of fucoidan, alginic acid and glucan from Turbinaria conoides by

sequential extraction with inorganic solvents

Plant material and preliminary treatments. Samples of Turbinaria conoides were collected

from Okha coast of Gujarat, India, in August 1995. The collected seaweeds were washed

thoroughly with tap water and dried by forced air circulation, followed by pulverization in a

Waring Blender.

Preparation of depigmented algal powder (DAP). After pulverization in a commercial grinder,

Depigmentation of algal powder (270 g) was done using sequential extraction with petroleum

ether (24 h) and acetone (24 h) as solvent in a Soxhlet apparatus. The unextracted material was

placed in a plastic beaker, and air dried to obtain depigmented algal powder (DAP, 173 g).

Extraction with acid. Extractions of DAP (3 g) with 0.1 M HCl (w/v::1:100) were conducted at

30–35°C for 12 h under constant stirring (twice). Separation of the residue from the extract was

performed by filtration through a glass filter (G-2). The residue was washed with additional 0.1

M HCl and the wash was collected to maximize polysaccharide recovery. The extracts were

combined, dialyzed and lyophilized to give fraction A (yield, 8.8%).

Extraction with alkali. The acid insoluble residue was extracted with 3% Na2CO3 (w/v::1:100) at

45–50°C for 4 h under constant stirring (twice). The extract was carefully acidified with

concentrated HCl to pH~1, the precipitate formed was collected by centrifugation, washed with

0.1 M HCl, suspended in water and dissolved by careful addition of NaOH. The slightly alkaline

solution was dialyzed and diluted with 4.0 M CaCl2 solution to make a final concentration of 2%

CaCl2. The precipitate formed at this stage was isolated by centrifugation, washed with water and

treated with 0.1 M HCl (4 x 50 ml, stirring at room temperature for ~2 h). Then the precipitate

was dissolved in dilute NaOH as above, the solution dialyzed and finally lyophilized to yield

sodium alginate (B, yield, 22.6%).

Extraction with water. The residue left after extraction with alkali was once again extracted with

water at 80°C (twice) and the combined extract (C) dialyzed. Then 4.0 M CaCl2 solution was

added to the extract to make a final concentration of 2% CaCl2, and the suspension kept at 5–9°C

overnight. After separating the precipitate (C2) by centrifugation the supernatant dialyzed,

56

concentrated and lyophilized to give fraction C1 (yield, 6.2%). The resulting insoluble residue

was dialyzed against distilled water and finally lyophilized (INS).

4-1.2.Isolation of polysaccharides from Adhatoda vasica

Plant material and preliminary treatments. Leaves of Adhatoda vasica were collected from the

garden of medicinal plants, The University of Burdwan, West Bengal, India. Collected leaves

(10 g) were washed thoroughly with tap water and then blended with water (800 ml) in a mixer

(Waring Products, Inc., Torrington, CT, USA).

Extraction of polysaccharides. Extraction of polysaccharide was conducted by stirring a

suspension of this paste in water (pH 6.0) at 25–32°C for 12 h. Separation of the residue from the

extract was performed by filtration through a glass filter (G-2). The insoluble material was

extracted twice more under similar condition at a solute to solvent ratio of 1:100 (w/v). The

combined liquid extract was dialyzed extensively against water and lyophilized. The recovered

material was dissolved in water, precipitated by the addition of ethanol (4 volumes) and then

collected by centrifugation (repeated three times). The final pellet was dissolved in water and

lyophilized to yield the water extracted polysaccharide, named WE (100 mg).

Isolation of arabinogalactan protein (AGP) with β-glucosyl Yariv reagent. AGP was isolated

according to Schultz et al., 2000. Briefly to a solution of WE in 1% NaCl (w/v) was added an

equal volume of β-glucosyl Yariv reagent also in 1% NaCl. The mixture was kept at 4°C for 18 h

and then centrifuged. The pellet was washed with 1% NaCl followed by pure methanol (3 times

each), dried and treated with sodium metabisulfite (10%). The resulting solution was dialyzed

and freeze dried to yield the arabinogalactan protein (AGP).

4-2. ANALYTICAL METHODS

4-2.1. General

All the experiments were carried out at least in duplicate, the mean and standard deviation

was directly calculated from the functions present in excel program. Evaporations were executed

under reduced pressure at around 45°C. Dialysis was performed against distilled water with

continuous stirring, toluene being added to inhibit microbial growth. During experiments,

moisture was determined by drying ground material in an air-circulated oven at 110°C for 3

hours. Chemicals used were analytical grade or best available. Except otherwise stated data

57

presented are the mean values of at least three independent analyses. When the standard

deviation was greater than 5%, experiments were repeated.

4-2.2. Sugar analysis

Total sugars were estimated by the phenol-sulphuric acid method (Dubois et al., 1956).

Evaluation was corrected afterwards, depending on sugar composition. Total uronic acids

present, were assayed using m-phenyl phenol color reagent (Ahmed & Labavitch, 1977). For

measurement of individual neutral sugar polysaccharides (10-12 mg) were hydrolyzed with 2 M

trifluoro acetic acid (2 h, 100°C).For the insoluble residues, this hydrolysis was followed by a

treatment with 72% (w/w) H2SO4 for 1 h at room temperature and after that, with 1 M H2SO4 for

2 h at 100°C. Myo-Inositol was used as internal standard, and hydrolytic losses were accounted

for by using an external standard (Chattopadhyay et al., 2007b). Monosaccharides were reduced

with sodium borohydride in anhydrous dimethyl sulfoxide and after reduction, the excess sodium

borohydride was decomposed by the addition of acetic acid. Acetylation was conducted by acetic

anhydride and 1-Methylimidazole was added as catalyst (Blakeney et al., 1983). Derived alditol

acetates were analysed by GC (Blakeney et al., 1983) on columns of 3% SP-2340 on

Supelcoport 100–120 mesh, and DB-225 (JW) and by GC-MS (Ghosh et al., 2009b; Ghosh et

al., 2010) or alternatively, trimethylsilyl (TMS) derivatives of methyl glycosides were analysed

by gas chromatography.

4-2.3. Protein estimation

Proteins present in soluble fractions were estimated by the method of Lowry et al., 1951 using

bovine serum albumin as standard. In the insoluble residues it was obtained by estimated the

total nitrogen and multiplying the value with 6.25.

Alternatively, protein was measured in the soluble material by the micro-Bradford method

using bovine serum albumin as standard, and was detected in some chromatography fractions by

the absorbance at 280 nm. Amino acids were released by hydrolysis with 6 M HCl at 110°C for

22 h in a sealed tube. Protections were done for cysteine, methionine and tyrosine using proper

protecting reagents. The liberated amino acids were analyzed by Pharmacia LKB ALPHA PLUS

amino acid analyzer.

58

4-2.4. Sulfate estimation

Turbidometric method. Samples (90 - 100 mg) were hydrolyzed in of 2 N hydrochloric acid (5

ml) in sealed glass tubes at 100oC (Chattopadhyay et al., 2010). Sulfate was then estimated using

a modified turbidometric barium chloride method (Craigie et al., 1984). The reagent was

prepared by dissolving 600 mg of gelatin in 200 ml of distilled water at 70oC and the solution

was allowed to cool. After 16 h at 4oC the temperature was brought to 30 – 35oC and 2.0 g of

BaCl2 .2H2O was added and then dissolved. AR grade K2SO4 was used here as a standard in the

range of 10 – 50 µg S ml-1. Accurately weighted samples (25-50 mg) were hydrolyzed for 2 h at

100oC with 2N HCl (0.75 ml) in a sealed tube. After that, the contents were transferred and made

to volume in a 10 ml volumetric flask. The humic substances were removed by centrifugation. 2

ml of sample, 18 ml of distilled water and 2 ml of HCl (0.5 N) was mixed in a 50 ml Erlenmeyer

flask. 1 ml of BaCl2-gelatin reagent was added to that and swirled. After 30 minutes swirling

again mixed the contents of the flasks and turbidity was measured at 550 nm against a reagent

blank.

Spectroscopic method. The sulfate content was measured according to the method of Rochas &

coworkers (1986) by considering the ratio of the intensity of the 1252 cm-1 band to the intensity

of the 2920 cm-1 band.

4-2.5. Desulfation

Desulfation of the purified fucoidan (AF3) obtained from T. conoides was carried out

according to the procedure of Falshaw & Furneaux (1998). The pyridinium salt of the sample (1

g) was dissolved in DMSO containing 10% MeOH, and then the solution was kept for 3 h at

100oC. The reaction mixture was cooled, diluted with an equal volume of water, and the pH

adjusted to 9.0 by the addition of sodium hydroxide (0.1 M). The solution was dialyzed against

running water for 20 h and after that lyophilization of the dialyzate gave desulfated sample.


Methylation was carried out using the method of Blakeney & Stone (1985) as described by

Adhikari & coworkers (2006). Exactly weighed (1-2mg) of the dried samples along with

magnetic bars were taken in tubes fitted with air tight rubber corks. The nitrogen atmosphere was

maintained within the tubes. The samples were dissolved in DMSO and to this solution, lithium

methyl carbanion were added. The mixture was then stirred in nitrogen atmosphere for 1 hr. The

tubes were cooled under ice bath and ice cold methyl iodide (0.4 ml) was added to it. After that

59

the tubes was left for 1 hr at room temperature. The methylation process was repeated once more

and excess methyl iodide was removed under vacuum. The resulting methylated polymer was

then extracted with chloroform-methanol [2:1 (v/v)]. The permethylated polysaccharide thus

obtained was hydrolyzed with trifluoro acetic acid (2.5 M) at 120°C for 75 min, reduced with

NaBD4(1 M) in NH4OH (2 M) for 3 h at room temperature and acetylated with acetic anhydride

using perchloric acid as a catalyst. The partially methylated alditol acetates (PMAA) were then

analyzed by GC and GC/MS as described by Sinha et al., 2010. The PMAA were identified by

(i) measurement of relative retention times (ii) methoxyl substitution pattern as obtained from

GC-MS and (iii) carbohydrate composition of the non methylated polymers.

4-2.7. Chromatography

Thin layer chromatography (TLC). The sugars released by acid hydrolysis (as described above)

were also analyzed by TLC as described by Ray, 2006. Briefly, TLC was then conducted by

ascending technique using silica gel G impregnated with 0.5M NaH2PO4 as the supporting

material. Small amounts of samples were spotted in the plate for identification. The Rf values of

the test material were compared to that with the 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.

The solvent system used for developing TLC was the following: (1) Ethanol: Phenol: Pyridine:

0.1 M Phosphoric acid :: 10 : 2 : 2 : 4 and (2) 2-Propanol: Methanol: Water :: 16 : 1 : 3

Detection: By spraying saturated solution of aniline phthalate and heating the plate at 100°C.

Size exclusion chromatography (SEC). SEC was performed as described by Chattopadhyay et

al., 2007a. In brief, solutions (1-2 ml) of different extracts in 0.5 M sodium acetate buffer (pH

5.0), were loaded to columns (i) (2.6 x 50 cm; Amersham Biosciences AB) of Superdex-30 (for

C1 solution of T. conoides), (ii) (90 cm x 2.6 cm, Amersham Biosciences AB) of Sephacryl S-

200 (for the solution of the fucoidan fraction (AF3) of T. conoides ) and (iii) (90 cm x 2.6 cm,

Amersham Biosciences AB) Sephacryl S-100 (for the solution of water extracted polymer (WE)

solution of A. vasica ) equilibrated with the same buffer. The column was then eluted

ascendingly with the same buffer at 0.5 ml min-1 at a temperature of 30–35°C. Elution of

polysaccharide was expressed as a function of the partition coefficient Kav [Kav = (Ve-V0)/(Vt-

V0) where, Vt and V0 are the total and void volume of the column determined as the elution

60

volume of glucose and blue dextran, respectively and Ve is the elution volume of the sample].

Here, the column was calibrated with standard dextrans.

Anion Exchange Chromatography (AEC). A solution (20 ml) of dilute acid extracted polymer

from T. conoides (fraction A) in 50 mM sodium acetate (pH 5.5) was applied to a column (2.6 x

25 cm) of DEAE-Sepharose FF (AcO-1). After that, the column was eluted (0.6 ml min-1)

successively with 0.05 M (fraction AF1), 0.7 M (fraction AF2) and 2.0 M (fraction AF3) NaOAc

buffer in a stepwise manner. Appropriate fractions were pooled, dialyzed and then lyophilized.

Gas chromatography (GC).GC was conducted on (i) GC-17A, Shimadzu and (ii) HP6890 series

Chromatograph.

The gas chromatograph was equipped with a flame ionization detector, the following columns

(a – d) and helium as gas vector.

(a) WCOT fused silica capillary column (length 25 m, i.d. 0.25 mm) and film thickness 0.4µm

with CP-Sil 5 CP as stationary phase and helium as gas vector, (b) 3 % SP-2340 on Supelcoport

100-120 mesh, (c) DB-225 (JW) and (d) SGE BP 225.

The oven temperature program was:

(a) 210°C isothermal or (b) 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 (c)170°C for 15min, 170-210°C at 5°C/min and 210°C for 15 min.

Gas liquid chromatography–Mass Spectrometry (GC-MS): The GC-MS analysis were conducted

using (a) Shimadzu (Japan) GC 17A coupled to QP 5050A MS and (b) GC (HP 6890) coupled to

an Autospec (Micromass, Manchester, UK) MS.

The mass spectra were recorded with MS instrument at 70 eV.

4-2.8. Spectroscopy

NMR Spectroscopy. The 1H NMR spectra of the polysaccharide containing fractions of T.

conoides were recorded on a Bruker 500 spectrometer (Bruker Biospin AG, Fallanden,

Switzerland) operating at 500 MHz, respectively, for 1H. The samples (~10 mg of each) were

heated at 80°C for 30 min with water (1 ml), centrifuged and the resulting supernatant

lyophilised. The freeze-dried sample was deuterium-exchanged by lyophilisation with D2O and

then examined in 99.9% D2O.

61

Scavenging effect (%) =

UV-VIS spectroscopy. UV-VIS spectra were recorded with Shimadzu UV- 1600A

spectrophotometer.

Infra-Red spectroscopy. IR spectra were obtained on a FT-IR spectrophotometer (JASCO FT/IR

- 420) using KBr discs containing finely ground samples.

Mass spectrometry. EI mass spectra were recorded using Shimadzu QP5050A GC/MS

instrument at 70 eV. The characterization of sugars, including 6-O-methyl galactose, was done

on the basis of previously reported mass spectra (Carpita & Shea, 1988) and relative retention

times as described by Ray & Lahaye, 1995.

4-3. BIOASSAY

4-3.1. FRAP assay

The FRAP assay was conducted according to Benzie & Strain, 1996 as modified by Pulido et

al., 2000. Briefly, the oxidant was prepared by mixing 2.5 ml of a 10 mM TPTZ [2,4,6-tri(2-

pyridyl-5-triazine) Fluka Chemicals, Madrid, Spain] solution in 40 mM HCl with 25 ml of 0.3 M

acetate buffer (pH 3.6) and 2.5 ml of 20 mM FeCl3.6H2O. The final solution has Fe(III) of 1.67

mM and TPTZ of 0.83 mM. To measure FRAP value, 900 µl of freshly prepared FRAP reagent

was warmed to 37°C and a reagent blank reading was taken at 593 nm; then 30 µl of test sample

and 90µl of distilled water were added. Absorbance readings were taken after 0.5 s and every 15

s until 30 min using a Shimadzu UV-1601 (PC) Spectrophotometer. The change of absorbance

(DA = A30 min_ A0min) was calculated and related to DA of an Fe(II) standard solution. Aqueous

solutions of known Fe(II) concentrations (100–2000 µM FeSO4.7H2O) were used for calibration.

4-3.2. Scavenging capability for 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals

The method reported by Shimada et al., 1992 was adopted for measurement of free radical

scavenging capability. To each 4 ml of sample solution, 1 ml of freshly prepared methanolic

DMSO solution of DPPH (0.5 mM) was added, mixed well and then let stand for 30 min at room

temperature in the dark. The absorbance of the resulting was recorded at 517 nm. Butylated

hydroxyanisole (BHA) was used as reference compound. The capability to scavenge the DPPH

radical was calculated using the following equation:

x100

62

4-3.3. Antitussive activity of the arabinogalactan from A. vasica

Animals. Animals used in this experiment were kept in faculty animal house, Department of

Experimental Pharmacology, Slovak Academy of Science, Dobra Voda, Slovakia, with food and

water ad libitum and with a standard air conditioning system. The animals were kept one week in

quarantine before starting the experiment. The experimental protocols were approved by the

Institutional Ethics Committee of the Jessenius Faculty of Medicine, Comenius University in

Martin, Slovakia, registered in Institutional Review Board/Institutional Ethic Board Office (IRB

00005636), complied with Slovakian and European Community regulations for the use of

laboratory animals and follow the criteria of experimental animal’s well fare.

Assessment of chemically induced cough and airways defense reflexes. Awaken guinea-pigs

were individually placed in a body plethysmograph box (HSE type 855, Hugo Sachs Elektronik,

Germany) and restricted so that the head protrudes into the nasal chamber and the neck were

sealed with a soft diaphragm. The cough reflex was induced by aerosol of citric acid in a

concentration 0.3 M. The citric acid aerosol was generated by a jet nebulizer (PARI jet nebulizer,

Paul Ritzau, Pari-Werk GmbH, Germany, output 5 l s−1, particles mass median diameter 1.2µm)

and delivered to the head chamber of the plethysmograph for 3min interval, in which number of

cough efforts was counted. The cough effort was defined as sudden PC-recorded enhancement of

expiratory flow associated with typical cough motion and sound followed by trained observer

(Sutovska et al., 2009a).The reactivity of the airway smooth muscle in vivo conditions was

expressed as values of specific airway resistance calculated according to Pennock et al., 1979 by

time difference between pressure changes in head and chest parts of body plethysmograph during

normal breathing pattern. Both, influence on citric acid-induced cough and specific airway

resistance were registered before any agent application (values labeled as N in graphs) and after

that in 30, 60, 120 and 300 min time intervals. The minimal time interval between two

measurements was 2 h to prevent cough receptors adaptation as well as adaptation of laboratory

animals on kind of irritation. All tested compounds (polysaccharide, codeine and vehicle) were

applied by peroral route of administration, plant polysaccharides in the dose of 50mgkg−1,

codeine in the dose 10 mg kg−1 and saline water (vehicle) in the dose 1ml kg−1 body weight.

Statistics. Student’s t-test was used for the statistical analysis of the obtained results. Data are

presented as mean±standard error of the mean (S.E.M.). p< 0.05 was considered statistically

63

significant. Significance of p < 0.05; p < 0.01 and p < 0.001 is shown by one, two or three

asterisks, respectively.

64

REFERENCES

Abd El-Megeed Hashem, F., & Ahmed Elsawi, S. (1998). Isoquinoline and quinazoline alkaloids of Adhatoda vasica NEES. Pharmaceutical and Pharmacological Letters, 8, 167-169.

Adachi, Y. S., Jinushi, T., Yadomae, T., & Ohno, N. (2002). Th1-oriented immunomodulating activity of gel-forming fungal (1-3)-beta-glucans. International journal of medicinal mushrooms, 4, 95–109.

Adhikari, U., Mateu, C. G., Chattopadhyay, K., Pujol, C. A., Damonte, E. B., & Ray, B. (2006). Structure and antiviral activity of sulphated fucans from Stoechospermum marginatum. Phytochemistry, 67, 2474–2482.

Aguirre, M. J., Isaacs, M., Matsuhiro, B., Mendoza, L., & Zúñiga, E. A. (2009). Characterization of a neutral polysaccharide with antioxidant capacity from red wine. Carbohydrate Research, 344, 1095-1101.

Ahmed, A., & Labavitch, J. M. (1977). A simplified method for accurate determination of cell wall uronide content. Journal of Food Biochemistry, 1, 361–365.

Alban, S., & Franz, G. (2001). Partial synthetic glucan sulfates as potential new antithrombotics: a review. Biomacromolecules, 2, 354-361.

Alban, S., Schauerte, A., & Franz, G. (2002). Anticoagulant sulfated polysaccharides. Part I. Synthesis and structure-activity relationships of new pullulan sulfates. Carbohydrate Polymers, 47, 267–276.

Amin, A. H., & Mehta, D. R. (1959). A bronchodilator alkaloid (vasicinone) from Adhatoda vasica Nees. Nature, 184, 1317.

Atal, C. K., (1980). Chemistry and pharmacology of vasicine – A new oxytocic and abortifacient. Indian Drugs, 15, 15–18.

Athukorala, Y., Lee, K. W., Kim, S. K., & Jeon, Y. J. (2007). Anticoagulant activity of marine green and brown algae collected from Jeju Island in Korea. Bioresource Technology, 98, 1711–1716.

Baek, S. H., Lee, J. G., Park, S. Y., Bae, O. N., Kim, D. H., & Park, J. H. (2010). Pectic polysaccharides from Panax ginseng as the antirotavirus principals in ginseng. Biomacromolecules, 11, 2044–2052.

Balzarini, J., & Van Damme, L. (2007). Microbicide drug candidates to prevent HIV infection. Lancet, 369, 787-797.

Bandyopadhyay, S. S., Ghosh, D., Micard, V., Sinha, S., Chatterjee, U. R., & Ray, B. (2012). Structure, fluorescence quenching and antioxidant activity of a carbohydrate polymer from Eugenia jambolana. International Journal of Biological Macromolecules, 51, 158-164.

Bandyopadhyay, S. S., Navid, M. H., Ghosh, T., Schnitzler, P., & Ray, B. (2011). Structural features and in vitro antiviral activities of sulfated polysaccharides from Sphacelaria indica. Phytochemistry, 72, 276-283.

Barahona, T., Chandía, N. P., Encinas, M. V., Matsuhiro, B., & Zúñiga, E. A. (2011). Antioxidant capacity of sulfated polysaccharides from seaweed: a kinetic approach. Food Hydrocolloids, 25, 529-535.

Beal, M. F. (2003). Mitochondria, oxidative damage, and inflammation in Parkinson's disease. Annals of the New York Academy of Sciences, 991, 120-131.

65

Belvisi, M. G., & Hele, D. J. (2009). Cough sensors III. Opioid and cannabinoid receptors on vagal sensory nerves. In F. Chung, & J. G. Widdicombe (Eds.), Pharmacology and therapeutics of cough. Berlin/Heidelberg: Springer-Verlag, 63-76.

Benzie, I. F. F., & Strain, J. J. (1996). The ferric reducing ability of plasma (FRAP) as a measure of ‘‘antioxidant power”: The FRAP assay. Analytical Biochemistry, 239, 70–76.

Berteau, O., & Mulloy, B. (2003). Sulfated fucans, fresh perspectives: structures, functions, and biological properties of sulfated fucans and an overview of enzymes active toward this class of polysaccharide. Glycobiology, 13, 29R-40R.

Blakeney, A. B., Harris, P., Henry, R. J., & Bruce, A. B. (1983). A simple rapid preparation of alditol acetates for monosaccharide analysis. Carbohydrate Research, 113, 291–299.

Blakeney, A. B., & Stone, B. A. (1985). Methylation of carbohydrates with lithium methylsulphinyl carbanion. Carbohydrate Research, 140, 319-324.

Boh, B., Berovic, M., Zhang, J., & Zhi-Bin, L. (2007). Ganoderma lucidum and its pharmaceutically active compounds. Biotechnology annual review, 13, 265-301.

Bohn, J. B., & BeMiller, J. N. (1995). (1→3)-β-d-Glucans as biological response modifiers: a review of structure-functional activity relationships. Carbohydrate Polymers, 28, 3–14.

Boisson-Vidal, C., Chaubet, F., Chevolot, L., Sinquin, C., Theveniaux, J., Millet, J., Sternberg, C., Mulloy, B., & Fischer, A. M. (2000). Relationship between antithrombotic activities of fucans and their structure. Drug Development Research, 51, 216–224.

Borchers, A. T., Keen, C. L., M. Gershwin, & M. E. (2004). Mushrooms, Tumors, and Immunity: An Update. Experimental Biology and Medicine, 229, 393-406.

Carpita, N. C., & Shea, E. M. (1988). Linkage structure of carbohydrates by gas chromatography mass spectrometry (GC-MS) of partially methylated alditol acetates. In Analysis of Carbohydrates by GC and MS; Bermann, C. J., McGinis, G. D., Eds.; CRC Press: BocaRaton, FL, 157-212.

Chan, G. C., Chan, W. K., & Sze, D. M. (2009). The effects of beta-glucan on human immune and cancer cells. Journal of hematology & oncology, 2, 25.

Chandia, N. P., & Matsuhiro, B. (2008).Characterization of a fucoidan from Lessonia vadosa (Phaeophyta) and its anticoagulant and elicitor properties. International Journal of Biological Macromolecules, 42, 235-240.

Chatterjee, U. R., Bandyopadhyay, S. S., Ghosh, D., Ghosal, P. K., & Ray, B. (2011). In vitro anti-oxidant activity, fluorescence quenching study and structural features of carbohydrate polymers from Phyllanthus emblica. International Journal of Biological Macromolecules, 49, 637– 642.

Chattopadhyay, K., Adhikari, U., Lerouge, P., & Ray, B. (2007a). Polysaccharides from Caulerpa racemosa: Purification and structural features. Carbohydrate Polymers, 68, 407.

Chattopadhyay, K., Mateu, C. G., Mandal, P., Pujol, C. A., Damonte, E. B., & Ray, B. (2007b). Galactan sulfate of Grateloupia indica: Isolation, structural features and anti-viral activity. Phytochemistry, 68, 2428-2435.

Chattopadhyay, K., Ghosh, T., Pujol, C. A., Carlucci, M. J., Damonte, E.B., & Ray, B. (2008). Polysaccharides from Gracilaria corticata: Sulfation, chemical characterization and anti-HSV activities. International Journal of Biological Macromolecules, 43, 346–351.

Chattopadhyay, N., Ghosh, T., Sinha, S., Chattopadhyay, K., Karmakar, P., & Ray, B. (2010). Polysaccharides from Turbinaria conoides: structural features and antioxidant capacity. Food Chemistry, 118, 823–829.

Chattopadhyay, N., Nosál’ová, G., Saha, S., Bandyopadhyay, S. S., Flešková, D., & Ray, B. (2011). Structural features and antitussive activity of water extracted polysaccharide from Adhatoda vasica. Carbohydrate Polymers, 83, 1970-1974.

66

Chaturvedi, R. K., & Beal, M. F. (2008). PPAR: a therapeutic target in Parkinson's disease. Journal of neurochemistry, 106, 506-518.

Chauvet, P., Bienvenu, J. G., Theoret, J. F., Latour, J. G., & Merhi, Y. (1999). Inhibition of platelet-neutrophil interactions by fucoidan reduces adhesion and vasoconstriction after acute arterial injury by angioplasty in pigs. Journal of cardiovascular pharmacology, 34, 597–603.

Cheshenko, N., & Herold, B. C. (2002). Glycoprotein B plays a predominant role in mediating herpes simplex virus type 2 attachment and is required for entry and cell-to-cell spread. The Journal of general virology, 83, 2247–2255.

Chevolot, L., Foucault, A., Chaubet, F., Kervarec, N., Sinquin, C., Fisher, A. M., & Boisson-Vidal, C. (1999). Further data on the structure of brown seaweed fucans: relationships with anticoagulant activity. Carbohydrate Research, 319, 154-165.

Chevolot, L., Mulloy, B., Ratiskol, J., Foucault, A., & Colliec-Jouault, S. (2001). A disaccharide repeat unit is the major structure in fucoidans from two species of brown algae. Carbohydrate Research, 330, 529-535.

Chihara, G., Hamuro, J., Maeda, Y.Y., Arai, Y., & Fukuoka, F. (1970). Fractionation and Purification of the Polysaccharides with Marked Antitumor Activity, Especially Lentinan, from Lentinus edodes (Berk.) Sing. (An Edible Mushroom).Cancer Research, 30, 2776-2781.

Chizhov, A. O., Dell, A., Morris, H. R., Haslam, S. M., McDowell, R. A., Shashkov, A. S., Nifant’ev, N. E., Khatuntseva, E. A., & Usov, A. I. (1999). A study of fucoidan from brown seaweed Chorda filum. Carbohydrate Research, 320, 108–119.

Chung, K. F. (2009). Clinical cough VI: The need for new therapies for cough: Disease specific and symptom-related anti-tussives. Handbook of Experimental Pharmacology, 187, 343–368.

Claeson, U. P., Malmfors, T., Wikman, G., & Bruhn, J. G. (2000). Adhatoda vasica: A critical review of ethnopharmacological and toxicological data. Journal of Ethnopharmacology, 72, 1–20.

Colak, E. (2008). New markers of oxidative damage to macromolecules. Journal of medical Biochemistry, 27, 1-16.

Copeland, R., Balasubramaniam, A, Tiwari, V., Zhang, F., Bridges, A., Linhardt, R. J, Shukla, D., & Liu, J. (2008).Using a 3-O-sulfated heparin octasaccharide to inhibit the entry of herpes simplex virus type 1. Biochemistry, 47, 5774–5783.

Cornford, C. S., Morgan, M., & Ridsdale, L. (1993). Why do mothers consult when their children cough? Family Practice, 10, 193–196.

Craigie, J. S., Wen, Z. C., & van der Meer, J. P. (1984). Interspecific, Intraspecific and Nutritionally-Determined Variations in the Composition of Agars from Gracilaria spp. Botanica Marina, XXVII, 55-61.

Cumashi, A., Ushakova, N. A., Preobrazhenskaya, M. E., D’Incecco, A., Piccoli, A., Totani, L., Tinari, N., et al. (2007). A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds. Glycobiology, 17, 541–552.

Daniel, R., Berteau, O., Chevolot, L., Varenne, A., Gareil, P., & Goasdoue, N. (2001). Regioselective desulfation of sulfated L-fucopyranoside by a new sulfoesterase from the marine mollusk Pecten maximus: application to the structural study of algal fucoidan (Ascophyllum nodosum). European journal of biochemistry / FEBS, 268, 5617-5626.

Daniel, R., Berteau, O., Jozefonvicz, J., & Goasdoue, N. (1999). Degradation of algal (Ascophyllum nodosum) fucoidan by an enzymatic activity contained in digestive glands of the marine mollusc Pecten maximus. Carbohydrate Research, 322(3-4), 291–297.

67

DeBelder, A. N. (1993). In ‘‘Industrial Gums’’,eds, Whistler R. L., BeMiller J. N., Academic Press, INC, 399.

Dhuley, J. N. (1999). Antitussive effect of A. vasica extract on mechanical and chemical stimulation-induced coughing in animals. Journal of Ethnopharmacology, 67, 361–365.

Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956).Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28, 350–366.

Edenharder, R., Leopold, C., & Kries, M. (1995). Modifying actions of solvent extracts from fruit and vegetable residues on 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoxaline (MeIQx) induced mutagenesis in Salmonella typhimurium TA 98. Mutation research, 341, 303-318.

Ekblad, M., Adamiak, B., Bergstrom, T., Johnstone, K. D., Karoli, T., Liu, L., Ferro, V., & Trybala, E. (2010). A highly lipophilic sulfated tetrasaccharide glycoside related to muparfostat (PI-88) exhibits virucidal activity against herpes simplex virus. Antiviral Research, 86, 196–203.

Ernst, E. (1998). Complementary therapies for asthma: What patient use? The Journal of Asthma: Official Journal of the Association for the Care of Asthma, 35, 667–671.

Falshaw, R., & Furneaux, R. H. (1998). Structural analysis of carrageenans from the tetrasporic stages of the red algae Gigartina lanceata and Gigartina chapmanii (Gigartinaceae, Rhodophyta). Carbohydrate Research, 307, 325–331.

Fan, L., Jiang, L., Xu, Y., Zhou, Y., Shen, Y., Xie, W., Long, Z., & Zhou, J. (2011). Synthesis and anticoagulant activity of sodium alginate sulfates. Carbohydrate Polymers, 83, 1797–1803.

Farias, E. H. C., Pomin, V. H., Valente, A. P., Nader, H. B., Rocha, H. A. O., & Mourao, P. A. S. (2008). A preponderantly 4-sulfated, 3-linked galactan from the green alga Codium isthmocladum. Glycobiology, 18, 250–259.

Farias, W. R. L., Valente, A. P., Pereira, M. S., & Mourao, P. A. S. (2000).Structure and anticoagulant activity of sulfated galactans. Isolation of a unique sulfated galactan from the red algae Botryocladiaoccidentalis and comparison of its anticoagulant action with that of sulfated galactans from invertebrates. Journal of Biological Chemistry, 275, 29299–29307.

Feizi, T. (2000). Carbohydrate-mediated recognition systems in innate immunity. Immunological Reviews, 173, 79-88.

Feyerabend, T. B., Li, J. P., Lindahl, U., & Rodewald, H. R. (2006). Heparan sulfate C5-epimerase is essential for heparin biosynthesis in mast cells. Nature Chemical Biology, 2,195-196.

Franz, G., Pauper, D., & Alban, S. (2000). In B. S. Paulsen (Ed.), Bioactive carbohydrate polymers, Dordrecht: Kluwer Academic Publishers, 47–58.

French, C. T., Irwin, R. S., & Fletcher, K. E. (2002). Evaluation of a cough-specific quality-of-life questionnaire. Chest, 121, 1123–1131.

Fujihara, M., & Nagumo, T. (1993). An influence of the structure of alginate on the chemotactic activity of macrophages and the antitumor activity. Carbohydrate Research, 243, 211–216.

Fujimoto, K., Tomonaga, M., & Goto, S. (2006). A case of recurrent ovarian cancer successfully treated with adoptive immunotherapy and lentinan. Anticancer Research, 26, 4015-4018.

Ghosh, T., Auerochs, S., Saha, S., Ray, B., & Marschall, M. (2010). Anti-cytomegaloviral activity of sulfated glucans generated from a commercial preparation of rice bran. Antiviral Chemistry & Chemotherapy, 21, 85–95.

Ghosh, T., Chattopadhyay, K., Marschall, M., Karmakar, P., Mandal, P., & Ray, B. (2009a). Focus on antivirally active sulfated polysaccharides: From structure–activity analysis to clinical evaluation. Glycobiology, 19, 2–15.

68

Ghosh, T., Pujol, C. A., Damonte, E. B., Sinha, S., & Ray, B. (2009b). Sulfated xylomannans from the red seaweed Sebdenia polydactyla: structural features, chemical modification and antiviral activity. Antiviral Chemistry & Chemotherapy, 19, 235–242.

Glauser, B. F., Rezende, R. M., Melo, F. R., Pereira, M. S., Francischetti, I. M. B., Monteiro, R. Q., Rezaie, A. R., & Mourao, P. A. S. (2009). Anticoagulant activity of a sulfated galactan: Serpin-independent effect and specific interaction with factor Xa. Journal of Thrombosis and Haemostasis, 102, 1183–1193.

Gordon, M., Bihari, B., Goolsby, E., Gorter, R., Guralnik, M., Mimura, T., et al. (1998). A placebo e controlled trial of immune modulator, lentinan, in HIV positive patients: a Phase I/II trial. Journal of Medicine, 29, 305-330.

Gordon, M., Guralnik, M., Kaneko, Y., Mimura, T., Goodgame, J., & Lang, W. (1995). Further clinical studies of curdlan sulfate (CRDS)-an anti-HIV agent. Journal of Medicine, 26, 97-131.

Grasdalen, H. (1983). High-field 1H NMR spectroscopy of alginate: Sequential structure and linkage conformations. Carbohydrate Research, 118, 255–260.

Gunay, N. S., & Linhardt, R. J. (1999). Heparinoids: Structure, Biological Activities and Therapeutic Applications. Planta Medica, 65, 301-306.

Guven, K. V., Ozsoy, Y., & Ulutin, O. N. (1991). Anticoagulant, fibrinolytic and antiaggregant activity of carrageenans and alginic acid. Botanica Marina, 34, 429-432.

Halliwell, B., & Gutteridge, J. M. (1995). The definition and measurement of antioxidants in biological systems. Free Radical Biology & Medicine, 18, 125-126.

Halliwell, B., & Gutteridge, J. M. C. (1984). Oxygen toxicity, oxygen radicals, transition metals and disease. Biochemical Journal, 219, 1–4.

Hannah, B. P., Heldwein, E. E., Bender, F. C., Cohen, G. H., & Eisenberg, R. J.(2007). Mutational evidence of internal fusion loops in herpes simplex virus glycoprotein B. Journal of virology, 81, 4858–4865.

Heinecke, J. W. (1997). Mechanisms of oxidative damage of low density lipoprotein in human atherosclerosis. Current Opinion in Lipidology, 8, 268-274.

Heldwein, E. E, Lou, H., Bender, F. C, Cohen, G. H. Eisenberg R. J, & Harrison SC. (2006).Crystal structure of glycoprotein B from herpes simplex virus 1. Science.313, 217–220.

Hensel, A., & Meier, K. (1999). Pectins and xyloglucans exhibit antimutagenic activities against nitroaromatic compounds. Planta Medica, 65, 395-399.

Herold, B. C., Visalli, R. J., Susmarski, N., Brandt, C. R., & Spear, P.G. (1994). Glycoprotein C-independent binding of herpes simplex virus to cells requires cell surface heparan sulphate and glycoprotein B. The Journal of general virology,75, 1211–1222.

Herold, B. C., WuDunn, D., Soltys, N., & Spear, P. G. (1991). Glycoprotein C of herpes simplex virus type 1 plays a principal role in the adsorption of virus to cells and in infectivity. Journal of virology, 65, 1090–1098.

Hileman, R. E., Fromm, J. R., Weiler, J. M., & Linhardt, R. J. (1998). Glycosaminoglycan-protein interactions: definition of consensus sites in glycosaminoglycan binding proteins. BioEssays, 20, 156-167.

Hirano, M., Kiyohara, H., & Yamada, H. (1994). Existence of a Rhamnogalacturonan II-like Region in Bioactive Pectins from Medicinal Herbs. Planta Medica, 60, 450-454.

Hobbs, C. R. (2004). Medicinal value of Turkey Tail fungus Trametes versicolor (L.:Fr.) Pilát (Aphyllophoromycetideae). International journal of medicinal mushrooms, 6,195–218.

69

Holst, O. (1999). In ‘‘Endotoxin in Health and Disease’’, eds, Brade H., Morrison D. C., Opal S., Vogel S., Marcel Dekker, Inc., New York, 115.

Hromadkova, Z., Paulsen, B. S., Polovka, M., Kostalova, Z., & Ebringerova, A. (2012). Structural features of two heteroxylan polysaccharide fractions from wheat bran with anti-complementary andantioxidant activities. Carbohydrate Polymers, DOI: http://dx.doi.org/10.1016/ j. carbpol. 2012.05.021.

Hu, T., Liu, D., Chen, Y., Wu, J., & Wang, S. (2010). Antioxidant activity of sulfated polysaccharide fractions extracted from Undaria pinnitafida in vitro. International Journal of Biological Macromolecules, 46, 193-198.

Huang, D., Ou, B., & Prior, R. L. (2005). The Chemistry behind antioxidant capacity assays. Journal of Agricultural and Food Chemistry, 53, 1841–1856.

Huq, M. E., Ikram, M., & Warsi, S. A. (1967). Chemical composition of Adhatoda vasica Linn. II. Pakistan Journal of Scientific and Industrial Research, 10, 224-225.

Imeson, A. P. (2000). In “Handbook of hydrocolloids”, ed, Phillips, G. O.;Williams, P. A, 87-102.

Inngjerdingen, K. T., Coulibaly, A.; Diallo, D., Michaelsen, T. E., & Paulsen, B. S. (2006). A complement fixing polysaccharide from Biophytum petersianum Klotzsch, a medicinal plant from Mali, West Africa. Biomacromolecules, 7, 48–53.

Inngjerdingen, K. T., Kiyohara, H., Matsumoto, T., Petersen, D., Michaelsen, T. E., Diallo, D., Inngjerdingen, M., Yamada, H., & Paulsen, B. S. (2007). An immunomodulating pectic polymer from Glinus oppositifolius. Phytochemistry, 68, 1046-1058.

Jain, M. P., & Sharma, V. K. (1982). Phytochemical Investigation of Roots of AdhatodaVasica. Planta Medica, 46, 250-250.

Jain, M. P., Koul, S. K., Dhar, K. L., & Atal, C. K. (1980). Novel nor-Harmal Alkaloid from Adhatoda-Vasica. Phytochemistry, 19, 1880-1882.

Jain, S. K. (2006). Superoxide dismutase overexpression and cellular oxidative damage in diabetes. A commentary on "Overexpression of mitochondrial superoxide dismutase in mice protects the retina from diabetes-induced oxidative stress". Free radical biology & medicine, 41, 1187-1190.

Je, J. Y., Park, P. J., Kim, E. K., Park, J. S., Yoon, H. D., Kim, K. R., et al., (2009). Antioxidant activity of enzymatic extracts from the brown seaweed Undariapinnatifida by electron spin resonance spectroscopy. LWT – Food Science and Technology, 42, 874–878.

Jiang, Y. H., Jiang, X. L., Wang, P., Mou, H. L., Hu, X. K., & Liu, S. Q. (2008). The antitumor and antioxidative activities of polysaccharides isolated from Isaria farinosa B05. Microbiological Research, 163, 424–430.

Kapoor, L. D. (1990). CRC handbook of ayurvedic medicinal plants. Boca Raton: CRC Press.

Karinaga, R., Mizu, M., Koumoto, K., Anada, T., Shinkai, S., Kimura, T., et al., (2004). First observation by fluorescence polarization of complexation between mRNA and the natural polysaccharide schizophyllan. Chem Biodivers, 1, 634–639.

Kariya, Y., Mulloy, B., Imai, K., Tominaga, A., Kaneko, T., Asari, A., et al., (2004). Isolation and partial characterization of fucan sulphates from the body wall of sea cucumber Stichopus japonicus and their ability to inhibit osteoclastogenesis. Carbohydrate Research, 339, 1339–1346.

Karmakar, P., Ghosh, T., Sinha, S., Saha, S., Mandal, P., Ghosal, P. K., & Ray, B. (2009). Polysaccharides from the brown seaweed Padina tetrastromatica: Characterization of a sulfated fucan. Carbohydrate Polymers, 78, 416–421.

70

Karmakar, P., Pujol, C.A., Damonte, E.B., Ghosh, T., & Ray, B. (2010). Polysaccharides from Padina tetrastromatica: Structural features, chemical modification and antiviral activity. Carbohydrate Polymers 80, 514–521.

Kataoka-Shirasugi, N., Ikuta, J., Kuroshima, A., & Misaki, A. (1994). Antitumor Activities and Immunochemical Properties of the Cell-wall Polysaccharides from Aureobasidium pullulans. Bioscience, biotechnology, and biochemistry, 58, 2145-2151.

Kawabata, H., Germain, R. S., Vuong, P. T., Nakamaki, T., Said, J. W., & Koeffler, H. P. (2000). Transferrin Receptor 2-a Supports Cell Growth Both in Iron-chelated Cultured Cells and in Vivo. Journal of Biological Chemistry, 275, 16618-16625.

Kennedy, J. F., & White, C. A. (1988). In Carbohydrate Chemistry (Kennedy J. F ed) Clarendon Press Oxford, 220.

Kiyohara, H., Cyong, J. C., &Yamada, H. (1989). Relationship between structure and activity of the “ramified” region in anti-complementary pectic polysaccharides from Angelica acutiloba kitagawa. Carbohydrate Research, 193, 201-214.

Kiyohara, H., Yamada, H., Takemoto, N., Kawamura, H., Komatsu, Y., & Oyama, T. (1993).Characterization of in vitro IL-2 production enhancing and anticomplementary pectic polysaccharides from kampo (Japanese herbal) medicine ‘Juzen-Taiho-To’. Phytotherapy Research, 7, 367-375.

Kleymann, G. (2005). Agents and strategies in development for improved management of herpes simplex virus infection and disease. Expert opinion on investigational drugs, 14, 135–161.

Kodama, N., Komuta, K., & Nanba, H. (2003).Effect of Maitake (Grifola frondosa) D-Fraction on the activation of NK cells in cancer patients. Journal of medicinal food, 6, 371-377.

Kogan, G., Šoltés, L., Stern, R., & Gemeiner, P. (2007). Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications. Biotechnology Letters, 29, 17-25.

Kubal J. V., & Gralen, N. (1948). Industrial Gums: Polysaccharides and Their Derivatives. Journal of Colloid Science, 3, 457-471.

Kumar, S. S., Kumar, Y., Khan, M. S., & Gupta, V. (2010). New antifungal steroids from Turbinaria conoides (J. Agardh) Kutzing. Natural Product Research, 24, 1481-1487.

Kumar, S. S., Kumar, Y., Khan, M. S., Anbu, J., & De Clercq, E. (2011). Antihistaminic and antiviral activities of steroids of Turbinaria conoides. Natural Product Research, 25, 723-729.

Laquerre, S., Argnani, R., Anderson, D. B., Zucchini, S., Manservigi, R., & Glorioso, J. C. (1998). Heparan sulfate proteoglycan binding by herpes simplex virus type 1 glycoproteins B and C, which differ in their contributions to virus attachment, penetration, and cell-to-cell spread. Journal of virology, 72, 6119– 6130.

Latge, J. P., Kobayashi, H., Debeaupuis, J. P., Diaquin, M., Sarfati, J., Wieruszeski, J.M., Parra, E, Bouchara, J. P., & Fournet, B. (1994). Chemical and immunological characterization of the extracellular galactomannan of Aspergillus fumigatus. Infection and Immunity, 62, 5424-5433.

Lee, S. H., Athukorala, Y., Lee, J. S., & Jeon, Y. J. (2008). Simple separation of anticoagulant sulfated galactan from red algae. Journal of Applied Physiology, 20, 1053–1059.

Lichtenthaler, F., & Mondel, S. (1997). Carbohydrates as Organic Raw Materials. Pure and Applied Chemistry, 69, 1853-1866.

Linhardt, R. J., & Toida, T. (1997). Heparin Oligosaccharides: New Analogues Development and Applications, in: Carbohydrate in Drug Designing, (Zbigniew, J. W., Karl, A. N., eds.), Marcel Dekker, Inc., New York, 277-341.

Liu, J., & Mori, A. (2006). Oxidative damage hypothesis of stress-associated aging acceleration:

71

neuroprotective effects of natural and nutritional antioxidants. Research Communications in Biological Psychology, Psychiatry & Neuroscience, 30-31, 103-119.

Lowry, O. H., Rosebrough, N. J., Lewsfarr, A., & Randall, R. J. (1951).Protein measurement with the folin reagent. Journal of Biological Chemistry, 193, 265–275.

Maeda, M., Totomu, U., Harada, N., Sekiguchi, M., & Hiraoka, A. (1991). Heparinoid-active sulphated polysaccharides fromMonostroma nitidum and their distribution in the chlorophyta. Phytochemistry, 30, 3611-3614.

Maehara, Y., Tsujitani, S., Saeki, H., Oki, E., Yoshinaga, K., Emi, Y., Morita, M., Kohnoe, S., Kakeji, Y., Yano, T., & Hideo Baba, H. (2012). Biological mechanism and clinical effect of protein-bound polysaccharide K (KRESTIN): review of development and future perspectives. Surgery Today, 42, 8–28.

Mandal, P., Mateu, C. G., Chattopadhyay, K., Pujol, C. A., Damonte, E. B., & Ray, B. (2007). Structural features and antiviral activity of sulphated fucans from the brown seaweed Cystoseira indica. Antiviral Chemistry and Chemotherapy, 18,153–162.

Mandal, P., Pujol, C. A., Carlucci, M. J., Chattopadhyay, K., Damonte, E. B., & Ray, B. (2008). Sulfated xylomannan of Scinaia hetai: Isolation, structural features and antiviral activity. Phytochemistry, 69, 2193–2199.

Mandal, P., Pujol, C. A., Damonte, E. B., Ghosh, T., & Ray, B. (2010). Xylans from Scinaia hatei: Structural features, sulfation and anti-HSV activity. International Journal of Biological Macromolecules, 46, 173–178.

Mantovani, M. S., Bellini, M. F., Angeli, J. P. F., Oliveira, R. J., Silva, A. F., & Ribeiro, L. R. (2008). beta-Glucans in promoting health: prevention against mutation and cancer. Mutation Research, 658, 154−161.

Mardberg, K., Trybala, E., Glorioso, J. C., & Bergstr¨om, T. (2001).Mutational analysis of the major heparin sulfate-binding domain of herpes simplex virus type 1 glycoprotein C. The Journal of general virology,82, 1941–1950.

Mardberg, K., Trybala, E., Tufaro, F., & Bergstr¨om, T. (2002). Herpes simplex virus glycoprotein C is necessary for efficient infection of chondroitin sulphate expressing gro2C cells. The Journal of general virology, 83, 291–300.

Maruyama, H., & Ikekawa, T. (2007). Immunomodulation and antitumoractivity of a mushroom product, proflamin, isolated from Flammulina velutipes (W. Curt:Fr.) Singer(Agaricomycetideae). International journal of medicinal mushrooms 9,109–122.

Maruyama, H., Tamauchi, H., Iizuka, M., & Nakano, T. (2006). The role of NK cells in antitumor activity of dietary fucoidan from Undaria pinnatifida sporophylls (Mekabu). Planta Medica, 72, 1415-1417.

Matsubara, K., Matsuura, Y., Bacic, A., Liao, M.L., Hori, K., & Miyazawa, K. (2001). Anticoagulant properties of a sulfated galactan preparation from a marine green alga, Codium cylindricum. International Journal of Biological Macromolecules, 28, 395–399.

Matsukawa, R., Dubinsky, Z., Kishimoto, E., Masaki, K., Masuda, Y., & Takeuchi, T. (1997).A comparison of screening methods for antioxidant activity in seaweeds. Journal of Applied Phycology, 9, 29–35.

Matsushima, H., Minoshima, H., Kawanami, H., Ikushima, Y., Nishizawa, M., Kawamukai, A., et al., (2005). Decomposition reaction of alginic acid using subcritical and supercritical water. Industrial and Engineering Chemistry Research, 44, 9626–9630.

May, C. D. (1990). Industrial pectins: Sources, production and applications. Carbohydrate Polymers, 12, 79-99.

72

Mayer, A. M. S., & Lehmann, V. K. B. (2000). Marine pharmacology in 1998: Marine compounds with antibacterial, anticoagulant, antifungal, anti-inflammatory, anthelmintic, antiplatelet, antiprotozoal and antiviral activities; with actions on the cardiovascular, endocrine, immune and nervous systems: And other miscellaneous mechanisms of action. Pharmacologist, 42, 62–69.

Mazumder, S., Morvan, C., Thakur, S., & Ray, B. (2004).Cell wall polysaccharides from Chalkumra (Benincasa hispida) fruit.Part I. Isolation and characterization of pectins. Journal of Agriculture and Food Chemistry, 52, 2356–2362.

Meer, W. (1980). In ‘‘Handbook of water soluble gums and resins’’, ed, Davidson R. I., McGraw Hill, New York, 81.

Mizuno, M., Minato, K., Ito, H., Kawade, M., Terai, H., & Tsuchida, H. (1999).Anti-tumor polysaccharide from the mycelium of liquid-cultured Agaricus blazei mill. Biochemistry & Molecular Biology International, 47, 707-714.

Moreira, P. I., Smith, M. A., Zhu, X., Honda, K., Lee, H. G., Aliev, G., & Perry, G. (2005). Oxidative damage and Alzheimer's disease: are antioxidant therapies useful? Drug News Perspect, 18, 13-19.

Mourao, P. A. S. (2004). Use of sulfated fucans as anticoagulantand antithrombotic agents: Future perspectives. Current Pharmaceutical Design, 10, 967–981.

Mourao, P. A. S., Pereira, M. S., Pavao, M. S. G., Mulloy, B., Tollefsen, D. M., Mowinckel, M. C., et al., (1996). Structure and anticoagulant activity of a fucosylated chondroitin sulphate from Echinoderm. Journal of Biological Chemistry, 271, 23973–23984.

Mukherjee, A. B., Zhang, Z., & Chilton, B. S. (2007). Uteroglobin: a steroid-inducible immunomodulatory protein that founded the Secretoglobin superfamily. Endocrine reviews, 28, 707-725.

Mulloy, B. (2005). The specificity of interactions between proteins and sulfated polysaccharides. Anais da Academia Brasileira de Ciências, 77, 651-664.

Mulloy, B., & Linhardt, R. J. (2001). Order out of complexity--protein structures that interact with heparin.Current opinion in structural biology, 11, 623-628.

Murugesan, S., Xie, J., & Linhardt, R. J. (2008). Immobilization of Heparin: Approaches and Applications. Current Topics in Medicinal Chemistry,8, 80-100.

Nanba, H., Hamaguchi, A., & Kuroda, H. (1987). The chemical structure of an antitumor polysaccharide in fruit bodies of Grifola frondosa (maitake).Chemical & pharmaceutical bulletin, (Tokyo), 35, 1162-1168.

Nasra, J., & Belvisi, M. G. (2009). Modulation of sensory nerve function and the cough reflex: Understanding disease pathogenesis. Pharmacology & Therapeutics, 124, 354–375.

Nishino, T., & Nagumo, T. (1991). The sulfate-content dependence of the anticoagulant activity of a fucan sulfate from the brown seaweed Ecklonia kurome. Carbohydrate Research, 214, 193-197.

Nishino, T., & Nagumo, T. (1992). Anticoagulant and antithrombin activities of oversulfated fucans. Carbohydrate Research, 229, 355-362.

Nishino, T., Nishioka, C., Ura, H., & Nagumo, T. (1994). Isolation and partial characterization of a novel amino sugar-containing fucan sulfate from commercial Fucus vesiculosus fucoidan. Carbohydrate Research, 255, 213-224.

Nishino, T., Yokoyama, G., Dobashi, K., Fujihara, M., & Nagumo, T. (1989). Isolation, purification, and characterization of fucose-containing sulfated polysaccharides from the brown seaweed Ecklonia kurome and their blood-anticoagulant activities. Carbohydrate Research, 186, 119-129.

73

Nosál’ová, G., Sutovska, M., Mokry´, J., Kardosˇová, A., Capek, P., & Khan, T. H. M. (2005). Efficacy of the herbal substances according to cough reflex. Minerva Biotechnologia, 17, 141–152.

Nosáľová, G., Prisenžňáková, Ľ., Košťálová, Z., Ebringerová, A., & Hromádková. Z., (2011).Suppressive effect of pectic polysaccharides from Cucurbita pepo L. var. Styriaca on citric acid-induced cough reflex in guinea pigs. Fitoterapia, 82, 357-364.

Nosáľová, G., Capek, P., Matáková, T., Nosáľ, S., Flešková, D., & Jureček, Ľ. (2012).Antitussive activity of an extracellular Rhodella grisea proteoglycan on the mechanically induced cough reflex. Carbohydrate Polymers, 87, 752-756.

Olafsdottir, E. S., Ingolfsdottir, K., Barsett, H., Paulsen, B. S., Jurcic, K., &Wagner, H. (1999a). Immunologically active (1→3)-(1→4)-α-D-Glucan from Cetraria islandica. Phytomedicine,6, 33-39.

Olafsdottir, E. S., Omarsdottir, S., Paulsen, B. S., Jurcic, K., & Wagner, H. (1999b). Rhamnopyranosylgalactofuranan, a new immunologically active polysaccharide from Thamnolia subuliformis. Phytomedicine, 6, 273-279.

Olafsdottir, E. S., Omarsdottir, S., Paulsen, B. S., & Wagner, H. (2003). Immunologically active O6-branched (1-->3)-beta-glucan from the lichen Thamnolia vermicularis var. subuliformis. Phytomedicine, 10, 318-324.

Olofsson, S., & Bergstrom, T. (2005). Glycoconjugate glycans as viral receptors. Annals of Medicine.37,154–172.

Ooi, V. E., & Liu, F. (2000). Immunomodulation and anti-cancer activity of polysaccharide-protein complexes.Current Medicinal Chemistry, 7, 715-729.

Ou, S. Y., Jackson, G. M., Jiao, X., Chen, J., Wu, J. Z., & Huang, X. S. (2007). Protection against oxidative stress in diabetic rats by wheat bran feruloyl oligosaccharides. Journal of Agricultural and Food Chemistry, 55, 3191–3195.

Painter, T. J. (1983). In: Aspinall G. O, editor. The polysaccharides, Academic Press Inc, 2, 195.

Patankar, M. S., Oehninger, S., Barnett, T., Williams, R. L., & Clark, G. F. (1993). A revised structure for fucoidan may explain some of its biological activities. The Journal of biological chemistry, 268, 21770-21776.

Paulsen, B., & Samuelsen, A. B. (2001). Plantago Species. Harwood Academic Publishers GmBH, Amsterdam.

Pavord, I. D. (2004). Cough and asthma. Pulmonary Pharmacology & Therapeutics, 17, 399–402.

Pavord, I. M., & Chung, K. F. (2008). Chronic cough: Treatment and management. The Lancet, 371, 1375–1384.

Paz-Elizur, T., Sevilya, Z., Leitner-Dagan, Y., Elinger, D., Roisman, L. C., & Livneh, Z. (2008). DNA repair of oxidative DNA damage in human carcinogenesis: potential application for cancer risk assessment and prevention. Cancer letters, 266, 60-72.

Pennock, B. E., Cox, C. P., Rogers, R. M., Cain, W. A., & Wells, J. H. (1979). A noninvasive technique for measurement of changes in specific airway resistance. Journal of Applied Physiology, 46, 399–406.

Pereira, M. S., Mulloy, B., & Mourao, P. A. S. (1999). Structure and anticoagulant activity of sulfated fucans: Comparison between the regular repetitive and linear fucansfrom echinoderms with the more heterogeneous and branched polymersfrom brown algae. Journal of Biological Chemistry, 274, 7656–7667.

Pereira, M. S., Silva, A. C. E. S. V., Valente, A. P., & Mourao, P. A. S. (2002). A 2-sulfated, 3-linked α-L-galactan is an anticoagulant polysaccharide. Carbohydrate Research, 337, 2231–2238.

74

Piculell, L. (1995). In ‘‘Food Polysaccharides and their Applications’’, ed, Stephen A. M., Marcel Dekker, Inc, Newyork, 2005.

Pomin, V. H., & Mourao, P. A. S. (2008). Structure, biology, evolution, and medical importance of sulfated fucans and galactans. Glycobiology, 18, 1016–1027.

Pomin, V. H., Pereira, M. S., Valente, A. P., Tollefsen, D. M., Pavao, M. S., & Mourao, P. A. S. (2005). Selective cleavage and anticoagulant activity of a sulfated fucan: stereospecific removal of a 2-sulfate ester from the polysaccharide by mild acid hydrolysis, preparation of oligosaccharides, and heparin cofactor II-dependent anticoagulant activity. Glycobiology, 15, 369-381.

Potin, P., Patier, P., Floc'h, J. Y., Yvin, J. C., Rochas, C., & Kloareg, B. (1992). Chemical characterization of cell-wall polysaccharides from tank-cultivated and wild plants of Delesseria sanguinea (Hudson) Lamouroux (Ceramiales, delesseriaceae): culture patterns and potent anticoagulant activity. Journal of Applied Phycology, 4, 119-128.

Pulido, R., Bravo, L., & Saura-Calixto, F. (2000).Antioxidant activity of dietary polyphenols as determined by a modified ferric reducing/antioxidant power assay. Journal of Agricultural and Food Chemistry, 48, 3396–3402.

Qi, H., Zhang, Q., Zhao, T., Chen, R., Zhang, H., Niu, X., & Li, Z. (2005). Antioxidant activity of different sulfate content derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta) in vitro. International Journal of Biological Macromolecules, 37, 195-199.

Qi, H., Zhang, Q., Zhao, T., Hu, R., Zhang, K., & Li, Z. (2006). In vitro antioxidant activity of acetylated and benzoylated derivatives of polysaccharide extracted from Ulva pertusa (Chlorophyta). Bioorganic & medicinal chemistry letters, 16, 2441-2445.

Quiocho F. A., & Vyas, N. K. (1999). In Bioorganic Chemistry: Carbohydrates (Hecht, S. M. edited), Oxford University Press, New York, 441-457.

Ramallo, I. A., Salazar, M. O., Mendez, L., & Furlan, R. L. E. (2011). Chemically Engineered Extracts: Source of Bioactive Compounds. Accounts of Chemical Research, 44, 241-250.

Ray, B. (2006). Polysaccharides from Enteromorpha compressa: isolation, purification and structural features. Carbohydrate Polymers, 66, 408-416.

Ray, B., & Lahaye, M. (1995). Cell-wall polysaccharides from the marine green algae Ulva rigida (Ulvales, Chlorophyta). Chemical structure of ulvan. Carbohydrate Research, 274, 313-318.

Rechter, S., K¨onig, T., Auerochs, S., Thulke, S., Walter, H., D¨ornenburg, H., Walter, C., & Marschall, M. (2006). Antiviral activity of arthrospira-derived spirulan-like substances. Antiviral research,72(3), 197–206.

Renn, D. W. (1997). Biotechnology and the red seaweed polysaccharide industry:Status, needs and prospects. TIBTECH, 15, 9–14.

Rocha de Souza, M. C., Marques, C. T., Dore, C. M. G., Ferreira da Silva, F. R., Rocha, H. A. O., & Leite, E. L. (2007). Antioxidant activities of sulphated polysaccharides from brown and red seaweeds.Journal of Applied Phycology, 19, 153–160.

Rochas, C., Lahaye, M., & Yaphe, W., (1986). Sulfate content of carrageenan and agar determined by infrared spectroscopy. Botanica Marina,29, 335-340.

Rojers, D. J., Jurd, K. M., Blunden, G., Paoletti, S., & Zanetti, F. (1990). Journal of Applied Physiology, 2, 357.

Rolin, C., & De Vries, J. (1990). In ‘‘Pectin, Food Gels’’, ed, P. Harris, Elsevier Applied Science, New York, 401.

Ruperez, P. (2001). Antioxidant activity of sulphated polysaccharides from the Spanish marine seaweed Nori. In Proceedings of the COST 916 European conference on bioactive compounds in plant

75

foods. Tenerife, Canary Islands, Spain: Health Effects and Perspectives for the Food Industry, 114.

Ruperez, P., Ahrazem, O., & Leal, A. (2002). Potential antioxidant capacity ofsulphated polysaccharides from the edible marine brown seaweed Fucus vesiculosus. Journal of Agricultural and Food Chemistry, 50, 840–845.

Rusnati, M., Vicenzi, E., Donalisio, M., Oreste, P., Landolfo, S., & Lembo, D. (2009). Sulfated K5 Escherichia coli polysaccharide derivatives: A novel class of candidate antiviral microbicides. Pharmacology and Therapeutics, 123, 310–322.

Rydberg, E., Westfall, M. J., Nicholas, R. A. (1999). Low Molecular weight heparin in preventing and treating DVT. American Family Physician, 59, 1607-1612.

Saha, S., Galhardi, L. C. F., Yamamoto, K. A., Linhares, R. E. C., Bandyopadhyay, S. S., Sinha, S., Nozawa, C., & Ray, B. (2010). Water-extracted polysaccharides from Azadirachta indica leaves: Structural features, chemical modification and anti-bovine herpesvirus type 1 (BoHV-1) activity. International Journal of Biological Macromolecules, 47, 640–645.

Saha, S., Navid, M. H., Bandyopadhyay, S. S., Schnitzler, P., & Ray, B. (2012). Sulfated Polysaccharides from Laminaria angustata: Structural features and in vitro antiviral activities. Carbohydrate polymers, 87, 123-130.

Saha, S., Nosál’ová, G., Ghosh, D., Flešková, D., Capek, P., & Ray, B. (2011). Structural features and in vivo antitussive activity of the water extracted polymer from Glycyrrhiza glabra. International Journal of Biological Macromolecules,48, 634-638.

Sahoo, D. (2010). Common Seaweeds of India. I. K. International Pvt Ltd, 102.

Saito, H., Ohki, T., & Sasaki, T. (1979). A 13C-nuclear magnetic resonance study of polysaccharide gels. Molecular architecture in the gels consisting of fungal, branched (1 → 3)-β-D-glucans (lentinan and schizophyllan) as manifested by conformational changes induced by sodium hydroxide. Carbohydrate Research, 74, 227-240.

Saito, H., Ohki, T., Takasuka, N., & Sasaki, T. (1977).A 13C-N.M.R.-Spectral study of a gel-forming, branched (1→3)-β-d-glucan, (lentinan) from lentinus edodes, and its acid-degraded fractions.Structure, and dependence of conformation on the molecular weight. Carbohydrate Research, 58, 293–305.

Sakurai, M. H., Matsumoto, T., Kiyohara, H., & Yamada, H. (1999). B-cell proliferation activity of pectic polysaccharide from a medicinal herb, the roots of Bupleurum falcatum L. and its structural requirement. Immunology, 973, 540-547.

Samuelsen, A. B., Paulsen, B. S., Wold, J. K., Knutsen, S. H., & Yamada, H. (1998). Characterization of a biologically active arabinogalactan from the leaves of Plantagomajor L. Carbohydrate Polymers, 35, 145-153.

Samuelsen, A. B., Paulsen, B. S., Wold, J. K., Otsuka, H., Kiyohara, H., Yamada, H., & Knutsen, S. H. (1996). Characterization of a biologically active pectin from Plantago major L. Carbohydrate Polymers,30, 37-44.

Sasaki, T., & Takasuka, N. (1976). Further study of the structure of lentinan, an anti-tumor polysaccharide from Lentinus edodes. Carbohydrate Research, 47, 99-104.

Schappert, S. M., & Burt, C. W. (2006). Ambulatory care visits to physician offices, hospital outpatient departments and emergency departments: United States, 2001–02. Vital and Health Statistics, 13, 1–66.

Schneider-Schaulies, J. (2000). Cellular receptors for viruses: Links to tropism and pathogenesis. The Journal of general virology,81, 1413–1429.

76

Schultz, C. R., Johnson, K. L., Currie, G., & Bacic, A. (2000).The classical arabinogalactan protein gene family of Arabidopsis. The Plant Cell, 12, 1751–1767.

Selby, H. H., & Whistler, R. L. (1993). In ‘‘Industrial Gums’’, ed, Whistler R. L., BeMiller J. N., Academic Press, INC, 87.

Sepulveda, R. T., & Watson, R. R. (2002). Treatment of antioxidant deficiencies in AIDS patients. Nutrition research. (N.Y.), 22, 27-37.

Sheu, J. H., & Sung, P. J. (1991). Isolation of 24-hydroperoxy-24-vinylcholesterol and fucosterol from the brown alga Turbinaria conoides. Journal of the Chinese Chemical Society, 38, 501-503.

Sheu, J. H., Wang, G. H., Sung, P. J., & Duh, C. Y. (1999).New cytotoxic oxygenated fucosterols from the brown alga Turbinaria conoides.Journal of Natural Products, 62, 224-227.

Shimada, K., Fujikawa, K., Yahara, K., & Nakamura, T. (1992). Antioxidative properties of xanthan on the autoxidation of soybean oil in cyclodextrin emulsion. Journal of Agricultural and Food Chemistry, 40, 945–948.

Shin, K. S., Kiyohara, H., Matsumoto, T., & Yamada, H. (1998).Rhamnogalacturonan II dimers cross-linked by borate diesters from the leaves of Panax ginseng C.A. Meyer are responsible for expression of their IL-6 production enhancing activities. Carbohydrate Research, 307, 97-106.

Silva, T. M. A., Alves, L. G., Queiroz, K. C. S., Santos, M. G. L., Marques, C. T., Chavante, S. F., Rocha, H. A. O., & Leite, E. L. (2005). Partial characterization and anticoagulant activity of a heterofucan from the brown seaweed Padina gymnospora. Brazilian Journal of Medical and Biological Research, 38, 523-533.

Simões, J. N., Nunes, F. M., Domingues, M. d. R. M., & Coimbra, M. A. (2010).Structural features of partially acetylated coffee galactomannans presenting immunostimulatory activity.Carbohydrate Polymers, 79, 397–402.

Singh, R. S., Misra, T. N., Pandey, H. S., & Singh, B. P. (1991).Aliphatic Hydroxyketones from Adhatoda-Vasica.Phytochemistry, 30, 3799-3801.

Sinha, S., Astani, A., Ghosh, T., Schnitzler, P., & Ray, B. (2010). Polysaccharides from Sargassum tenerrimum: Structural features, chemical modification and antiviral activity. Phytochemistry, 71, 235-42.

Sinha, S. Bandyopadhyay, S. S., Ghosh, D., Chatterjee, U. R., Saha, S., Ghosal, P. K., & Ray, B. (2011a). Structural Characteristics, Fluorescence Quenching, and Antioxidant Activity of the Arabinogalactan Protein-rich Fraction from Senna (Cassia angustifolia) Leaves. Food Science Biotechnology, 20, 1005-1011.

Sinha, S., Nosal’ova, G., Bandyopadhyaya, S. S., Fleˇskovab, D., & Ray, B., (2011b). In vivo anti-tussive activity and structural features of a polysaccharide fraction from water extracted Withania somnifera. Journal of Ethnopharmacology, 134, 510–513.

Spear, P.G. (2004). Herpes simplex virus: Receptors and ligands for cell entry. Cellular microbiology,6, 401–410.

Springer, G. F., Wurzel, H. A., McNeal, Jr. G. M., Ansell, N. J., & Doughty, M. F. (1957).Isolation of anticoagulant fractions from crude fucoidan. Proceedings of the Society for Experimental Biology and Medicine. 94, 404–409.

Stone, B. A., &Clarke, A. E. (1992). Chemistry and biology of (1,3)-D-glucans. Victoria, Australia: La Trobe University Press; 882.

Sultana, N., Anwar, M. A., Ali, Y., & Afza, N. (2005). Phytochemical studies on Adhatoda vasica. Pakistan Journal of Scientific and Industrial Research, 48, 180-183.

77

Sutovska, M., Nosal’ova, G., Fraˇnova, S., & Kardoˇsova, A. (2007). Isolation, characterization, and antitussive activity of polysaccharides from the flowers of Althaea officinalis L., var Robusta, leaves of Arctium lappa L., var. Herkules, and gum exutade of peach tree (Prunus persica L., Batsch). Bratislava Medical Journal, 108, 93–99.

Sutovska, M., Fraňová, S., Prisežnaková, L., Nosáľová, G., Togola, A., Diallo, D., Paulsen, B. S., & Capek, P., (2009a).Antitussive activity of polysaccharides isolated from the Malian medicinal plants, International Journal of Biological Macromolecules, 44, 236-239.

Sutovska, M., Nosáľová, G., Šutovský, J., Fraňová, S., Prisenžňáková, Ľ., & Capek, P. (2009b). Possible mechanisms of dose-dependent cough suppressive effect of Althaea officinalis rhamnogalacturonan in guinea pigs test system. International Journal of Biological Macromolecules, 45, 27-32.

Sutovska, M., Fraňová, S., Sadloňová, V.,Grønhaug, T. E.,Diallo, D.,Paulsen, B. S., & Capek, P. (2010). The relationship between dose-dependent antitussive and bronchodilatory effects of Opilia celtidifolia polysaccharide and nitric oxide in guinea pigs. International Journal of Biological Macromolecules, 47, 508-513.

Sutovska, M., Capek, P., Fraňová, S., Pawlaczyk, I., & Gancarz, R. (2012).Antitussive and bronchodilatory effects of Lythrum salicariapolysaccharide-polyphenolic conjugate. International Journal of Biological Macromolecules, 51, 794-799.

Sutovska, M., Capek, P., Kocmálová, M., Fraňová, S., Pawlaczyk, I., & Gancarz, R. (2013). Characterization and biological activity of Solidago canadensis complex. International Journal of Biological Macromolecules, 52, 192-197.

Synytsya, A., Kim, W. J., Kim, S. M., Pohl, R., Synytsya, A., Kvasnička, F., Čopíková, J., & Park, Y. I. (2010). Structure and antitumour activity of fucoidan isolated from sporophyll of Korean brown seaweed Undaria pinnatifida. Carbohydrate Polymers, 81, 41-48.

Tal-Singer, R., Peng, C., Ponce de Leon, M., Abrams, W. R., Banfield, B. W., Tufaro, F., Cohen, G. H., & Eisenberg, R. J. (1995). Interaction of herpes simplex virus glycoprotein gC with mammalian cell surface molecules. Journal of virology, 69, 4471– 4483.

Taylor, K. R., & Gallo, R. L. (2006). Glycosaminoglycans and their proteoglycans: host-associated molecular patterns for initiation and modulation of inflammation.The FASEB Journal, 20, 9-22.

Thakur, M., Weng, A., Fuchs, H., Sharma, V., Bhargava, C. S., Chauhan, N. S., Dixit, V. K., & Bhargava.S. (2012). Rasayana properties of Ayurvedic herbs: Are polysaccharides a major contributor. Carbohydrate Polymers, 87, 3-15.

Thappa, R. K., Agarwal, S. G., Dhar, K. L., Gupta, V. K., & Goswami, K. N. (1996). Two pyrroloquinazolines from Adhatoda vasica. Phytochemistry, 42, 1485-1488.

Therkelsen, G. H. (1993). In ‘Industrial Gums: Polysaccharides and Their Derivatives, edited by James N. BeMiller, Roy L. Whistler, Academic Press Inc, 7, 146-180.

Tolstoguzov, V., (2004). Why are polysaccharides necessary? Food Hydrocolloids, 18, 873-877.

Tomoda, M., Ohara, N., Shizimu, N., & Gonda, R. (1994a). Characterization of a novel glucan, which exhibits reticuloendothelial system-potentiating and anti-complementary activities, from the rhizome of Cnidium officinale. Chemical & pharmaceutical bulletin, 42, 630-633.

Tomoda, M., Ohara, N., Shizimu, N., & Gonda, R. (1994b). Characterization of a novel heteroglucan from the rhizome of Cnidium officinale exhibiting high reticuloendothelial system-potentiating and anti-complementary activities. Biological & pharmaceutical bulletin, 17, 973-976.

78

Trybala, E., Bergstr¨om, T., Svennerholm, B., Jeansson, S., Glorioso, J. C., & Olofsson, S. (1994). Localization of a functional site on herpes simplex virus type 1 glycoprotein C involved in binding to cell surface heparan sulphate. The Journal of general virology, 75, 743–752.

Tseng C. K. (1946). In ‘‘Colloid Chemistry’’, J. Alexander, ed, Rheinhold Publishing corp, New York, 6, 630.

Tsuchida, H. M., Taniguchi, Y., Ito, H., Kawade, M., & Akasaka, K. (2001). Glucomannan separated from Agaricus blazei mushroom culture and antitumor agent containing as active ingredient. In J. Patent (Ed.) (Vol. 11–080206).

Unursaikhan, S., Xu, X., Zeng, F., & Zhang, L. (2006). Antitumor activities of O-sulfonated derivatives of (1-->3)-alpha-D-glucan from different Lentinus edodes. Bioscience, biotechnology, and biochemistry, 70, 38-46.

Usui, T. (1980). Isolation of highly purified fucoidan from Eisenia bicyclics and its anticoagulant and antitumor activities. Agricultural and biological chemistry, 44, 1965-1966.

Van de Perre, P. (2003). Transfer of antibody via mother's milk. Vaccine, 21, 3374-3376.

Voragen, A. G. J., Pilnik, W., Thibault, J. F., Axelos, M. A. V., & Renard, C. M. G. C. (1995). In: Food polysaccharides and their applications, Stephen A M. (Eds), Marcel Dekker, Inc., New York, 287-339.

Wagner, H., & Jordan, E. (1988). An immunologically active arabinogalactan from Viscum album ‘berries’. Phytochemistry, 27, 2511-2517.

Wang, B. G., Zhang, W.W., Duan, X. J., & Li, X. M. (2009a). In vitro antioxidative activities of extract and semi-purified fractions of the marine red alga, Rhodomela confervoides (Rhodomelaceae).Food Chemistry, 113, 1101–1105.

Wang, J., Liu, L., Zhang, Q., Zhang, Z., Qi, H., & Li, P. (2009b). Synthesized oversulphated, acetylated and benzoylated derivatives of fucoidan extracted from Laminaria japonica and their potential antioxidant activity in vitro. Food Chemistry, 114, 1285–1290.

Wang, J., Zhang, Q., Zhang, Z., & Li, Z. (2008). Antioxidant activity of sulphated polysaccharide fractions extracted from Laminaria japonica. International Journal of Biological Macromolecules, 42, 127–132.

Wang, J., Zhang, Q., Zhang, Z., Song, H., & Li, P. (2010). Potential antioxidant and anticoagulant capacity of low molecular weight fucoidan fractions extracted from Laminaria japonica. International Journal of Biological Macromolecules, 46, 6-12.

Wasser, S. P. (2002). Medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides. Applied microbiology and biotechnology, 60, 258-274.

Wealth of India, (1988). A dictionary of Indian raw materials: Volume II: B (revised). New Delhi: Publications and Information Directorate. (350 + 90 pp. indexes).

Wealth of India, (1985). Algae, CSIR, New Delhi, Vol-IA, 138.

Whistler, R. (Ed.) (1993). Exudate gums. New York: Academic Press.

Witezak, Z. J. (1995). Carbohydrates as Drug and Potential Therapeutics. Current Medicinal Chemistry, 1, 392-405.

Witvrouw, M., & De Clercq, E. (1997). Sulfated polysaccharides extracted from sea algae as potential antiviral drugs. General Pharmacology, 29, 497–511.

Yamada, H., Ra, K. S., Kiyohara, H., Cyong, J. C., & Otsuka, Y. (1989a). Structural characterization of an anti-complementary pectic polysaccharide from the roots of Bupleurum falcatum L. Carbohydrate Research, 189, 209-226.

79

Yamada, H, Ra, K. S., Kiyohara, H., Cyong, J. C., Yang, H. C., & Otsuka,Y. (1989b). Characterization of anti-complementary neutral polysaccharides from the roots of Bupleurum falcatum. Phytochemistry, 27, 3163-3168.

Yamada, H., Kiyohara, H., Takemoto, N., Zhao, J. F., Kawamura, H., Komatsu, Y., Cyong, J. C., & Hosoya, E. (1992). Mitogenic and Complement Activating Activities of the Herbal Components of Juzen-Taiho-To1. Planta Medica, 58, 166-170.

Yamada, H., & Knutsen, S. H. (1996). Characterization of a biologically active pectin from Plantago major L. Carbohydrate Polymers, 30, 37-44.

Yamaguchi, T., Takamura, H., Matoba, T., & Terao, J. (1998). HPLC method for evaluation of the free radical scavenging activity of foods by using 1, 1,-diphenyl-2-picrylhydrazyl.Bioscience Biotechnology and Biochemistry, 62,1201–1204.

Yang, Y., Liu, D., Wu, J., Chen, Y., & Wang, S. (2011). In vitro antioxidant activities of sulfated polysaccharide fractions extracted from Corallina officinalis. International Journal of Biological Macromolecules, 49, 1031-1037.

Ye, H., Wang, K., Zhou, C., Liu, J., & Zeng, X. (2008). Purification, antitumor and antioxidant activities in vitro of polysaccharides from the brown seaweed Sargassum pallidum. Food Chemistry, 111, 428–432.

Yu, K. W., Kiyohara, H., Matsumoto, T., Yang, H. C., & Yamada, H. (1998). Intestinal Immune System Modulating Polysaccharides from Rhizomes of Atractylodes lancea. Planta Medica, 64, 714-719.

Yuan, X. P., Wang, J., Yao, H. Y., & Chen, F. (2005). Free radical scavenging capacity and inhibitory activity on rat erythrocyte hemolysis of feruloyl oligosaccharides from wheat bran insoluble dietary fiber. LWT – Food Science and Technology, 38, 877–883.

Zhang, L., Li, X., Xu, X., & Zeng, F. (2005). Correlation between antitumor activity, molecular weight, and conformation of lentinan. Carbohydrate Research, 340, 1515-1521.

Zhang, Y., Li, S., Wang, X., Zhang, L., & Cheung, P. C. K. (2011). Advances in lentinan: Isolation, structure, chain conformation and bioactivities. Food Hydrocolloids, 25, 196-206.

Zhao, J. F., Kiyohara, H., Matsumoto, T., & Yamada, H. (1994). Anti-complementary acidic polysaccharides from roots of Lithospermum euchromum. Phytochemistry, 34, 719-724.

Zhao, X., Xue, C., Cai, Y., Wang, D., & Fang, Y. (2005). Study of antioxidant activities of fucoidan from Laminaria japonica. High Technology Letters, 11, 91-94.

Zhou, G., Sun, Y., Xin, H., Zhang, Y., Li, Z., & Xu, Z. (2004). In vivo antitumor and immunomodulation activities of different molecular weight lambda-carrageenans from Chondrus ocellatus. Pharmacological research, 50, 47-53.

Zhou, S., & Gao Y. (2002). The immunomodulating effects of Ganoderma lucidum (Curt.:Fr.) P.Karst (LingZhi, Reishi Mushroom) (Aphylloromycetidae). International journal of medicinal mushrooms,4,1–11.

Zhu, H., Di, H., Zhang, Y., Zhang, J., & Chen, D. (2009). A protein-bound polysaccharide from the stem bark of Eucommia ulmoides and its anti-complementary effect. Carbohydrate Research, 344, 1319-1324.

Zhuang, C. Itoh, H., Mizuno, T., & Ito, H. (1995). Antitumor Active Fucoidan from the Brown Seaweed, Umitoranoo (Sargassum thunbergii). Bioscience, biotechnology, and biochemistry, 59, 563-567.

Zhuang, C., Mizuno, T., Ito, H., Shimura, K., Sumiya,T., Kawade, M., & Inamori, Y. (1994). Fractionation and Antitumor Activity of Polysaccharides from Grifola frondosa Mycelium.Bioscience, biotechnology, and biochemistry, 58, 185-188.

Zjawiony, J. K. (2004). Biologically active compounds from Aphyllophorales (polypore) fungi. Journal of natural products, 67, 300-310.

80

The novel findings presented in this thesis are:

1. Three families of water soluble polysaccharides, namely, sulfated fucan, alginic acid and

glucan can be easily isolated from the marine alga Turbinaria conoides.

2. A 50 kDa sulfated fucan containing inter alia, α-(1→3)-linked fucopyranosyl residues with a

sulfate group at the position 4, has been identified.

3. The alginic acid contains equimolar proportions of guluronic and mannuronic acid residues.

The guluronic acid content (G %) and G–G diad frequency (GG %) of this polymer are 46.2 and

42.1, respectively.

4. A 5 kDa linear glucan containing β-(1→3)-linked glucopyranosyl residue has also been

isolated.

5. These polymers T. conoides from demonstrated dose dependent antioxidative activity. The

fucoidan showed the highest antioxidative activity followed by the alginate and glucan.

6. The decoction of Adhatoda vasica contains a highly branched arabinogalactan.

7. This branched arabinogalactan containing 1,3- and 1,3,6-linked galactopyranosyl & 1,5- and

1,3,5- linked arabinofuranosyl residue, possesses potent in vivo antitussive activity.

8. Peroral administration of this arabinogalactan (50 mg kg−1 body weight) from A. vasica

inhibited the number of coughs induced by citric acid in guinea pigs and slightly decreased the

values of specific airway resistance.

9. Thus, traditional aqueous extraction methods provide polysaccharides, which stimulate an

intense biological response. This could represent an interesting approach in phytotherapeutic

treatments.

SUMMARY

81

LIST OF PUBLICATIONS

Nabanita Chattopadhyay, Tuhin Ghosh, Sharmistha Sinha, Kausik Chattopadhyay, Paramita

Karmakar, Bimalendu Ray (2010). Polysaccharides from Turbinaria conoides: Structural

features and antioxidant capacity. Food Chemistry, 118, 823–829.

Nabanita Chattopadhyay, Gabriella Nosal’ova, Sudipta Saha, Shruti S. Bandyopadhyay, Dana

Fleˇskova, Bimalendu Ray (2011). Structural features and antitussive activity of water extracted

polysaccharide from Adhatoda vasica. Carbohydrate Polymer, 83, 1970-1974.

POLYSACCHARIDES FROM Turbinaria conoides AND...

Documents

Transcript of POLYSACCHARIDES FROM Turbinaria conoides AND...