A PROJECT REPORT ON

REVIEW ON ISOLATION OF WATER SOLUBLE AND WATER INSOLUBLE POLYSACCHARIDES

BACHELOR OF PHARMACY

 

SUBMITTED BY 

MD SALIK

(BPH/1026/2011)

UNDER THE GUIDANCE OF 

Dr. K. JAYARAM KUMAR

(ASSOCIATE PROFESSOR)

DEPARTMENT OF PHARMACEUTICAL SCIENCES AND TECHNOLOGY

BIRLA INSTITUTE OF TECHNOLOGY

MESRA, RANCHI-835215

2015

CONTENTS

CHAPTER 1: INTRODUCTION

CHAPTER 2: ISOLATION OF WATER SOLUBLE NON-STARCH POLYSACCHARIDES

CHAPTER 3: ISOLATION OF WATER INSOLUBLE NON-STARCH POLYSACCHARIDES

CHAPTER 4: CONCLUSION

REFERENCES

INTRODUCTION

Polysaccharides are polymeric carbohydrates molecules composed of long chains of monosaccharide units. They are isolated from terrestrial and marine plants or are principally the exogenous metabolites of some bacteria.

Polysaccharides are composed of many monosaccharide residues that are joined one to the other by O-glycosidic linkages.

When all the monosaccharide in a polysaccharide are the same type the polysaccharide is called a homopolysaccharide or homoglycan, but when more than one type of monosaccharide is present they are called heteropolysaccharide or heteroglycan.

Classification of polysaccharides

Polysaccharides by source (Reddy, mohan et al .2011)

Seaweed extracts: Agars, aliginates, carrageenans

Higher plant cell wall insoluble: cellulose Higher plant cell wall soluble : pectin

Higher plant seeds: cereal starch, guar gum, locust bean gum  

Higher plant tuber & roots: potato starch, tapioca starch

Polysaccharides by structure  

Linear: amylose, cellulose,pectin,alginates  

Short branched: guar gum, locust beangum, Xanthan gum  

Branched-on-branch: amylopectin, gum arabic, arabinoxylan

Polysaccharides by monomers

Homoglycans: starch, cellulose  

Diheteroglycans: agars, alginate,carrageenans  

Triheteroglycans: Xanthan, gellan, arabinoglycan

Polysaccharides by charge  

Neutral: amylose,amylopectin, cellulose, guar gum

  Anionic: Alginates, carrageenans, gellan.

  Cationic: Chitosan

Types of Polysaccharides

Various examples of water soluble polysaccharides are Guar Gum, Dextran(Sluka Peter et al,2004), Gum Karaya, Heparin etc

Gum Karaya

It is the dried exudation of the Sterculia Urens Tree and other species of Sterculia It yields galactose, rhamnose, and galcturonic acid oh hydrolysis The major use of Gum Karaya is as a bulk laxative in view of its ability to form a

mucilaginous gel on contact with water

Guar Gum Guar gum is a galactomannan, which occurs as a storage polysaccharides consisting

of ( 1→4)- diequatorically linked β-D- mannose monomers,some of which are linked to single sugar side chains of α-D-galactose attached (Doyle et al ,2008).

Guar gum is particularly useful for colon delivery because it can be degraded by specific enzymes in this region of the gastrointestinal.

Structure of Guar gum

Dextran Dextran is a complex, branched glucan composed of chains of varying length It is used medicinally as an antiplatelet to reduce blood viscosity The straight chains consists of α-1,6 glycosidic linkages between glucose molecules,while

branching begin from α-1,3 linkages Dextan was first discovered by Louis Pasteur as a microbial product in wine

Structure of Dextran.

Water insoluble polysaccharides Starch Starch is a storage carbohydrate consisting of glucose monomers in plants such as cereals, root

vegetables and legumes It is comprised of two polymers, namely amylose and amylopectin. Most common cereal starches contain 15-30% amylose

Pectin

Pectin is a structural polysaccharide heteropolysaccharide contained in the primary cell walls of terrestrial plants.

It was first isolated and described in 1825 by Henri Bracannot It is used in food as a gelling agent,particularly in jams and jellies

Cellulose

Cellulose is a polysaccharide consisting of a linear chain of several hundred to many thousands of β(1→4) linked D-glucoseunits.

Cellulose is the most abundant organic polymer on Earth Cellulose is mainly used to produce paperboard and paper. Cellulose for industrial use is mainly obtained from wood pulp and cotton.

ISOLATION OF WATER SOLUBLE POLYSACCHARIDES

Water soluble polysaccharides are isolated from various parts of the plants mainly fruits, roots, pulp , seeds and leaf using a hot water extraction regime followed by ethanolic precipitation

Generally for isolation of water soluble polysaccharides raw materials are coarsely grounded and boiled/washed with hot water for different time periods

Generally cold centrifugation is done at 4 ᵒC- 8 ᵒC at 8000 rpm for 30 min. After centrifugation the supernatant is stored at 4 ᵒC in a freezer for at least 12 hours Supernatant is precipitated using alcohol/acetone Precipitated polysaccharides are removed by centrifugation. For further purification the crude polysaccharide is subjected to dialysis, followed by Gel

Permeation Chromatography/ Size exclusion chromatography using different chromatographic column.

Extraction of water soluble non starch polysaccharides from plants

Name of author

Name of Plant Plant part used Method of extraction Reagents used

Tao et al (2012)

Polygala tenifolia Roots The dried P tenifolia was refluxed in a

soxhlet apparatus with toluene-ethanol for

6 hours. Then it was filtered,concentrated

and cooled down. Then 95% ethanol was

added slowly until the concentration is

50% and kept for 1 hour. Then calcium

chloride was added and kept overnight to

precipitate tannin. The supernatant was

obtained by centrifugation and

ultrafiltration was done.

Toluene-ethanol

95% ethanol

CaCl2

Name of author

Name of Plant Plant part used

Method of extraction Reagents used

Huang et al (2012)

Cassia obtusifolia Seeds C.obtusifolia was purchased from the

market and grounded. Approx 5 gm of

finely ground powder samples were stirred

in distilled water and followed by boiling

it. After filtration water soluble

polysaccharide was precipitated with 95%

ethanol and collected by centrifugation.

Then the precipitates was dissolved in

water and dialysed to remove the small

molecules. The dialysed solution was

freeze dried to yield polysaccharide

powder

95% ethanol0.05 N NaOH

Name of author

Name of Plant Plant part used

Method of extraction Reagents used

Xie et al (2015)

Ampelopsismegalophylla

Leaves Dried leaves of A.megalophylla were

chopped into small pieces and mixed with

95% ethanol and then extracted with

distilled water. The aqueous extract was

filtered and polysaccharide were

precipitated by 95% ethanol , then

keeping it overnight and the precipitated

material were collected by

centrifugation. The extracts were then

deproteinised . Finally the precipitate was

washed with absolute ethanol, acetone

and ether and crude polysaccharide of

A.megalophylla was obtained

95% ethanol

Acetone

Ether

ISOLATION OF WATER INSOLUBLE POLYSACCHARIDES Water insoluble polysaccharides are divided into two groups e.g. Starch and Non-starch

polysaccharides Starch is a storage plant polysaccharide. It is isolated using aqueous extraction method or

through chemical treatment

Schematic representation of isolation

of starch

Non-starch water insoluble polysaccharides are cellulose, linin, hemicellulose etc. These 

water insoluble polysaccharides are soluble in organic solvents e.g. methanol, ethanol

Schematic representation of isolation of water insoluble non-starch polysaccharides

Extraction process of Water Insoluble Polysaccharides

Name of author

Name of Plant Plant part used Method of extraction Reagents used

Peng et al. (2004)

Ganoderma tsugae

FruitsMycelia of G .tsugae were obtained through

submerged cultivation and extraction. After

being defatted 100g mycelia were extracted

with 0.2 M phosphate buffer and the residue

was extracted with 2% NaOH. The

supernatants were neutralized with acetic acid

Both the precipitates after neutralization

were re-purified and finally after vacuum

dried for 7 days the white powder coded as

GM5-1 for the water insoluble polysaccharide

were extracted

0.2 M Phosphate buffer2 % NaOH

Name of author

Name of Plant Plant part used

Method of extraction Reagents used

Wang et al. (2012)

Panax japonicus RhizomeP. japonicus rhizomes were refluxed with

85% ethanol in a water bath. After

incubation, mixtures were centrifuged.

The insoluble residue was dried in an

oven and extracted. After elute was

concentrated in vacuum four times the

volume of ethanol was added to the

water and kept overnight in a

refrigerator to precipitate polysaccharide.

The precipitate was separated by

centrifugation and air dried

0.1 M NaOH85% ethanol

Name of author

Name of Plant Plant part used

Method of extraction Reagents used

Ding et al. (2011)

Solanum nigrum L SeedsCoarsely powdered of dried SNL was pre

extracted twice with 95% ethanol and

then refluxed twice with 80%

ethanol.The degreased powder were

dried.The aqueous extract was filtered and

the residue was extracted with 0.5 M

NaOH solution.The extraction was

condensed and precipitated with 95%

EtOH to obtain the crude polysaccharide

0.5 M NaOH

95 % EtOH

CONCLUSIONS Conclusion of this review can be divided into two parts

Isolation of water soluble polysaccharides Isolation of water insoluble polysaccharides

Isolation of water soluble polysaccharides

In general isolation of water soluble polysaccharides is done through precipitation method. This method has several steps. Among these steps boiling time, pre-precipitation centrifugation, polysaccharide precipitation and post-precipitation centrifugation are needed to be optimized for each sample. Petroleum ether, EtOH, n-BuOH are used for the isolation of water soluble polysaccharides.Among other methods alcohol precipitation method is most effective because of its recycle flexibility.

Isolation of water insoluble polysaccharides

Isolation of water insoluble polysaccharides is done through solvent extraction method. This method is needed to needed to be optimized for each sample. Starch is isolated either by simple aqueous extraction method or chemical treatment method. NaOH and 0.5 M KOH are widely used for the isolation of water insoluble polysaccharides.

REFERENCES

Xin, T., Zhang, F., Jiang, Q, Chen, C, Huang, D., Li, Y., Shen, W. & Jin, Y., (2012) Extraction, purification and antitumor activity of a water-soluble polysaccharide from the roots of Polygala tenuifolia . Carbohydrate Polymers, 90,1127– 1131

Huang, Y., Chaw, C., Hsiang, T. Y. (2012). Composition, characteristics, and in-vitro physiological effects of the water-soluble polysaccharides from Cassia seed. Food Chemistry, 134, 1967-1972.

Peng, Y., Zhang, L., Zhang, Y., Xu, X., Kennedy, J.F (2004) .Solution properties of water-insoluble polysaccharides from the mycelium of Ganoderma tsugae .Carbohydrate Polymers 59 (2005) 351–356

Wang, R., Chen, P.,Jia, F., Tang, J,. Ma, F., Xu, B (2012) . Characterization and antioxidant activities of polysaccharides from Panax japonicus C.A. Meyer .Carbohydrate Polymers 88 (2012) 1402– 1406

Ding,X., Zhu, F,. Gao, S (2011) .Purification, antitumour and immunomodulatory activity of water-extractable and alkali-extractable polysaccharides from Solanum nigrum L. Food Chemistry 131 (2012) 677–684

Dong, Q., Yao, J., Fang, Ji (2003) . Structural characterization of the water-extractable polysaccharides from Sophora subprostrata roots . Carbohydrate Polymers 54 (2003) 13–19

Xin,T., Zhang, F., Jiang, Q., Chen, C., Huang, D., Li, Y., Shen, W., Jin.,Y (2012) . Extraction, purification and antitumor activity of a water-soluble polysaccharide from the roots of Polygala tenuifolia . Carbohydrate Polymers 90 (2012) 1127– 1131

Palacios ,I., Garcia- Lafuente, A., Guillamon, E., Villares, A (2012) .Novel isolation of water-soluble polysaccharides from the fruiting bodies of Pleurotus ostreatus mushroom . Carbohydrate Research 358 (2012) 72-77

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Liu, C., Jiang, H., Zhou, H., Li, P., Wang, F (2015). Isolation, structural characterization and neurotrophic activity of a polysaccharide from Phellinus ribis .Carbohydrate Polymers 127 (2015) 145–151