EXTRACTION AND CHARACTERIZATION OF XYLOGLUCAN …

www.wjpps.com Vol 5, Issue 6, 2016.

2209

Bashir et al. World Journal of Pharmacy and Pharmaceutical Sciences

EXTRACTION AND CHARACTERIZATION OF XYLOGLUCAN

(TAMARIND SEED POLYSACCHARIDE) AS PHARMACEUTICAL

EXCIPIENT

Abeer Bashir*, Pramod Kumar Sharma and Musarrat Husain Warsi

Department of Pharmacy, School of Medical and Allied Sciences, Galgotias University,

Greater Noida, U.P. India-203201.

ABSTRACT

Recent study includes extraction and characterization of the xyloglucan

from tamarind (Tamarindus indica) seed polysaccharide as a

pharmaceutical agent. Tamarind seed polysaccharide was obtained by

water based extraction in Soxhlet apparatus. For characterization of the

extracted tamarind seed polysaccharide phytochemical screening was

done and micromeritic properties, flow behavior and swelling index

were determined. It was also found that extracted tamarind seed

polysaccharide had good flow properties and pH was 6.4, this showed

that it can be used in dosage form, without any irritation.

Mucoadhesive nature of extracted tamarind seed polysaccharide was

also evaluated by texture analyzer in different concentration range

(0.5% and 1%, w/v) and results revealed that it shows concentration based mucoadhesive

strength. It can be concluded that tamarind derived seed polysaccharide (xyloglucan) can be

used as pharmaceutical agent to prepare different types of formulations.

KEYWORDS: Tamarindus indica, Mucilage, Xyloglucan, Natural polysaccharide,

Mucoadhesion.

1. INTRODUCTION

In the recent years, plant derived polymers having mucilage which occur in high

concentrations with different parameters of interest. It have diverse pharmaceutical

applications as a diluents, binders, disintegrates in solid dosage forms, thickeners in oral

liquid dosage formulations, protective colloids in suspensions, gelling agents in gels and

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.041

Volume 5, Issue 6, 2209-2220 Research Article ISSN 2278 – 4357

*Corresponding Author

Abeer Bashir

Department of Pharmacy,

School of Medical and

Allied Sciences, Galgotias

University, Greater Noida,

U.P. India-203201.

Article Received on

21 April 2016,

Revised on 12 May 2016,

Accepted on 31 May 2016

DOI: 10.20959/wjpps20166-7021


2210


bases in suppository which enables them to participate for the formation of different dosage

forms.[1]

Natural gums and polymers when get in contact with the water, form gel layer on the

surface of the system and control the release of drug.[2]

Various natural gums come under the category of polysaccharide such as tragacanth, karaya,

acacia and khaya. Natural gums have been widely used as tablet, binders, emulgents and

thickeners in cosmetics and suspensions as film-forming agents and transitional colloids.

Generally most of the gums absorbs water, causes the gums to swell and exude by the

incision. Almost gums composed of salts such as calcium, magnesium and potassium salts of

bassoric acid, known as bassorin. Some of the gums have been reportedly used in an

insoluable powder for suspending as a suspending agent, an emulsifying agent in emulsion

for oils, resin and a binding agent.[3]

Tamarind seed polysaccharide is a natural polymer

which is derived from the tamarind seed. The main component of tamarind seeds has been

identified as a non-ionic, neutral, branched polysaccharide consisting of cellulose like

backbone that carries xylose and galactoxylose substituents. The configuration of (TSP)

tamarind seed polysaccharide gives the product a ‘mucin like’ molecular structure, thus

conferring optimal mucoadhesive properties. It has been also shown that, at the

concentrations present in the ophthalmic preparations under studied, one major feature of

TSP that makes it familiar to natural tears, i.e. its ability to crystallize in a fern-likee shape. It

has been suggested that due to the structure similarity of the TSP to endogeneous mucin may

allow formulations containing this polymer to adhere readily to the ocular surface for

prolonged periods and provide sustained release.[4]

Tamarind is a common tree of India and

Southeast Asia, which belongs to dicotyledonous family Leguminosae. A mucoadhesive

polymer extract obtained from the seed kernel of Tamarindus indica, possesses high

viscosity, broad pH tolerance, non‑carcinogenicity, mucoadhesive property and

biocompatibility.[7,8]

Tamarind seed polysaccharide (TSP) isolated from tamarind kernel

powder shows sustained release behavior for both water‑soluble (acetaminophen, caffeine,

theophylline and salicylic acid) and water insoluble (indomethacin) drugs.[9]

In addition to

this TSP have shown high drug holding capacity[10]

and high thermal stability.[11]

This has led

to its application as excipient in hydrophilic drug delivery system. A number of

mucoadhesive formulations have been developed using TSP for drugs such as gentamycin,

ofloxacin,[12]

paclitaxel,[13]

ketotifen fumarate[14]

with improved efficacy. The present study

was therefore aimed at to evaluate TSP (xyloglucan) as an pharmaceutical agent based on

different parameters.


2211


2. MATERIALS AND METHODS

2.1Extraction procedure

Tamarind seeds were purchased from the seeds shop situated in the old Delhi, India. Seeds

were boiled and mucilage was taken out to extract xyloglucan. Mucilage was dried, powdered

and further passed from sieve # 20 and stored in air tight container until used.

Extraction of tamarind polysaccharide includes two steps:

Step 1: Extraction of tamarind polysaccharide

Xyloglucan extracted by tamarind seeds were done as following

Crude tamarind seeds were washed neat and clean then kept for 3 hrs boiling. After

completion of boiling, boiled seeds were kept for cooling for 24 hrs so that mucilage is being

expelled out from the seeds. Completing 24 hrs of expulsion of mucilage from seeds, heat

them for few minutes then set for cooling. Pour seeds with mucilage in the muslin cloth and

press till all the mucilage is squeezed from the seeds. Temperature of extraction media was

maintained at 70 C and duration of extraction was adjusted about 6 hrs.[5,6]

Step 2: Isolation of tamarind seed polysaccharide

In hot water boiled tamarind seeds were kept for 24 hrs so that the mucilage can come out

from the seeds. After 24 hrs, boiled the seeds containing xyloglucan for 1 hr. Water extracted

juice were squeezed with muslin cloth bag and the concentrated juice was cooled to 4 C.

Tamarind seed polysaccharide xyloglucan was precipitated by alcohol-juice treatment

2:1(v/v) followed by continuous stirring for 15 min and mixture was further allowed to stand

for 2 hrs for better tamarind seed polysaccharide precipitation. This allows filtering of

polysaccharide substances because tamarind seed polysaccharide remains float on the surface

of alcohol-water mixture. Floating tamarind polysaccharide coagulate was filtered through

muslin cloth, washed with alcohol (95%) and squeezed. Squeezed tamarind seed

polysaccharide was further dried to constant weight at 35-45°C in hot air oven. Hard

tamarind seed polysaccharide cake was ground and sieved through sieve # 20, stored in

desiccators for further use.[5,6]


2212


Fig. 1: Configuration of Xyloglucan adopted from Manchanda et al., (2014)

3. Physicochemical characterization of Xyloglucan consisted in Tamarind Seed

Polysaccharide

3.1 Identification tests for carbohydrates

Extracted mucilage was mixed with Molish’s reagent followed by addition of sulfuric

acid.[14,15]

3.2 Determination of Purity of Xyloglucan in Tamarind Seed Polysaccharide

For determination of purity of extracted xyloglucan test for alkaloids, proteins, gum. Fats,

tannins and amino acids were performed.[14,15]

3.3 Solubility behavior

One part of dry tamarind seed polysaccharide powder was shaken with different solvents and

their solubility was determined. Solubility of tamarind seed polysaccharide was determined in

basic solvents i.e Acetone, Chloroform, Hexane, Butanol and Water. It has been observed

that polysaccharide is soluable in every solvent.[14]

3.4 pH of Xyloglucan in Tamarind Seed Polysaccharide:

Firstly, extracted Xyloglucan was weighed and then dissolved in water separately to get a 1%

w/v solution. The pH of solution was determined using pH meter.[14]

3.4 Swelling Index

A pre-weighed amount (100 mg) of powder was placed in simulated Intestinal fluid (SIF) (pH

7.4) and allowed to swell up to a constant weight. The powder were removed and blotted with

filter paper, and their changes in weight were measured. The swelling index was determined

by formula.[15]

Swelling index= X 100


2213


4. Powder properties

The flow properties of the sample were investigated by the angle of repose, bulk density and

tapped density measurements.[16]

The angle of repose was determined on 50 g of sample

using Borosilicate glass funnel (with orifice diameter and a base diameter of 6.5 mm and 8

cm respectively). Bulk and tapped densities were measured in 100 ml of graduated cylinder.

The sample contained in the cylinder was tapped mechanically by means of Auto-Tap Shaker

.The tapped volume was noted down when it showed no change in its value. The data

generated from bulk densities and tapped densities were used in figured the Carr’s index and

Hausner’s ratio for the xyloglucan powder.[16]

True density was obtained by helium

pycnometer. The data generated from bulk density and true densities were utilized for

calculating the porosity of powder mass. Moisture content was determined by official method

of USP32-NF27 in a mechanical convection oven at 105 C to the weight constants.

4.1 True density

Among various methods available for the determination of true density, liquid displacement

method is the one of frequent used method and was used in the present study. Acetone was

selected as the liquid for displacement because tamarind seed polysaccharide is soluble in

acetone. This method has been used by authors.[14,17,18]

4.2 Powder Compressibility (Carr’s index)

This property is also known as compressibility, finely tamarind seed polysaccharide 5g was

transferred into measuring cylinder and calculations were done using bulk density

apparatus.[14,17,18]

4.3 Particle size and particle distribution

TSP were first examined under optical microscope. After the optimization, the mean size

(surface average particle diameter) and size distribution of the extracted TSP were further

analyzed by a Hydro 2000MU Malvern particle size analyzer. TSP powders were suspended

in distilled water (1% w/v), sonicated for 60 sec prior to particle size determination.

Polydispersity was calculated by the following formula:

Polydispersity = (D0.9- D0.1) /D0.5

Where D0.9, D0.5 and D0.1 are the particle diameters determined at the 90th

,50th and 10

th

percentile of undersized particles, respectively.


2214


5. FT-IR Study

The FTIR spectral analysis of TSP was carried out by KBR pellet technique using FTIR

spectrophotometer of Alpha Bruker. The FTIR spectral analysis was scanned between 500 –

4000 cm-1

.

6. Mucoadhesion study

The mucoadhesion strength of the different concentration (0.5%. 0.75% and 1%, w/v) of TSP

was analyzed using TA.XT Plus Texture analyzer (Stable Micro Systems, Surrey, UK). The

adhesive tape was positioned on the tip of load cell and solution was placed on excised goat

cornea. Formulation was then applied under the load cell with cornea and 0.08 N force was

applied by the load cell for around 14 min. After this the tip was pulled back and force

required to remove the TSP from the cornea was calculated, which determine the

mucoadhesive strength. Control was taken 0.9%, w/v NaCl solution.

7. RESULT AND DISCUSSION

It has been observed that isolated tamarind seed polysaccharide was pinkish brown in color.

It consists no odor and taste along with irregular shape as well as hard and rough in sensation

and texture. The pH of 1% solution of TSP was found 6.4, which indicated that it should be

non-irritating for mucus membrane. Solubility study showed that it was sparingly soluble in

cold water, freely soluble and form viscous colloidal solution in warm water. Swelling index

of isolated Xyloglucan in tamarind seed polysaccharide was found to 660±0.50% . Swelling

index result revealed that the high swelling ability of the xyloglucan. High swelling of

polymer delays drug release up to desired time period. So it can be used in gel formulations

for controlled drug delivery.

The isolated sample of TSP was subjected to identification. The violet color ring appeared at

junction of mixture in test tube that confirms the presence of carbohydrates in sample

powder. Confirmation test was also carried out for tamarind seed polysaccharide which gave

positive test for mucilage, gums whereas negative for tannins, alkaloids and proteins. Other

phyto-constituents were absent in the isolated powder. This can be considered as proof for

purity of the isolated tamarind seed polysaccharide as depicted in table 3.

7.1 Percentage Yield

The % yield of the polysaccharide was found to be 20.0% for tamarind. During the

processing of tamarind xyloglucan isolation washing is required many times which may


2215


result in loss of dissolved polysaccharides. However extraction processes tamarind is easy

and hence gives better yield.

7.2 Solubility in various solvents

The solubility was checked in common solvents depending on their polarity such as water,

phosphate buffers pH6.8, 0.1N HCl, acetone, ethanol, methanol etc. The polysaccharide was

found to be soluble inorganic solvents and is also soluble in the aqueous solvents and swells

to make a viscous solution. Hence the polysaccharides are hydrophilic in nature.

7.3 Particle size and size distribution

The mean particle size of 1%, w/v TSP solution was 29.547 μm and the values of d(0.1),

d(0.5) and d(0.9) and polydispersity were found to be 5.180µm, 12.148µm, 29.547 µm and

1.357 respectively.

Fig. 1 Particle size report of 1%, w/v TSP Solution

7.4 Powder Properties

The polysaccharides were subjected to the analysis of powder flow properties such as bulk

density, tap density, Carr’s index (CI) and Hausner ratio (HR). Direct compression of

powders requires when materials show good flowability, comapactibility and compressibility.

These constraints become more decisive when the formulation contains large amount of

active substances with poor compression properties. Wet granulation method is selected for

production of porous and free-flowing granules, which enables to form tablets with high

mechanical strength at low compression pressure. The %CI range in between 26-31 stands for

poor flow. Again HR ranges in between 1.35-1.45 also possess poor flow. From the

investigation the values were found to be in this range for all three polysaccharides. Hence

they are of poor flow properties. The probable reason may be due to the high intermolecular


2216


force of attraction among the particles. It may be due to inadequate freeze drying of the

sample resulting fluffy powder which are tough to be sieved. The results are shown in Table

2.

Table: 1. Powder characteristics and flow properties of xyloglucan powder

Values Values

Angle of repose(degree) 27.35±0.12

Comptressibility index(%CI) 4.25±0.150

Hausner’s ratio(H) 1.03±0.06

Bulk density(ρo) 0.63±0.03

Tapped density(ρ) 0.749±0.016

Mean particle size (µm) 81.63±42.16

True density 0.83± 0.04

% porosity 41.67±2.34

% moisture content 4.31±0.78

Ash value (%) 6.23±0.251

Table: 2 Determination of purity of isolated tamarind seed polysaccharide

Tests Present/Absent

Carbohydrates +

Hexose Sugar +

Monosaccharides -

Proteins -

Fats and oils -

Alkaloides -

Amino acids -

Mucilage -

Gums -

+ Present; - Absent

7.5 Rheological Behaviour

The viscosities of the different concentration (0.5%, 0.75% and 1%, w/v) of TSP solutions

were determined by Brookfield viscometer at different shear. All the solutions showed

sufficient viscosity at different shear rate and shows pseudoplastic flow. This was observed

that viscosity decreases correspondingly with increasing shear. TSP shows consistent change

behavior upon application of pressure.

7.6 FTIR Spectrum

The IR spectrum of TSP is shown in the Fig.2 and its structural assignments are described in

table 3. Different stretch and bend vibrations showed their respective peaks. Different

functional groups were observed in IR spectra which are summarized in Table 3.


2217


Fig. 2 FT-IR spectroscopy of Tamarind Seed Polysaccharide

Table 3: Functional groups and peaks observed in IR spectra data of Xyloglucan

S. No Wave number (cm-1

) Functional group

1 3261.85 -OH stretch

2 2921.29-2852.68 Broad C-H str

3 1367.43 CH2 bend

4 1245.08-1207.47 -OH plane bend

5 1150.19 Glycosidic C-O-C stretch

6 998.41,940.49, 893.26 CH bend

7.7 Mucoadhesion Study

Result obtained by performing texture analyser (Fig. 3) revealed that the force needed to

detach the 0.9% NaCl solution was zero. This indicates the non-adhesive nature of control

solution. While in the case of TSP solution (1%, w/v) 0.07 N force was obtained which

demonstrated significant adhesion.

Fig.3 Texture analysis graphs (A) 0.9% NaCl Sol. (B) 0.5% w/v TSP Sol. (C) 0.75% w/v

TSP Sol. (D) 1.0% w/v TSP Sol.


2218


8. CONCLUSION

The polysaccharide was isolated and characterized in terms of various physicochemical

parameters of powder. From these characterized parameters it was observed that they possess

substantial viscosity and very good mucoadhesive property. The mucoadhesive character of

TSP may be due to resemblance with mucin structure. The other properties like pH,

solubility, flow property, bulk density etc. also found to be satisfactory. The observations

showed that TSP exhibited good compactability, cohesiveness and would be able to produce

better flow properties in its granular form. It may be concluded that these polysaccharide can

be chosen for the development of mucoadhesive drug delivery system as a carrier materials.

They may be utilized for the colon targeting, mucoadhesive nanoparticulate drug delivery,

nasal drug delivery, ocular drug delivery etc. for better result. The properties also make them

suitable for use in oral solid dosage formulation for sustained drug delivery action.

ACKNOWLEDGEMENT

Authors are highly thankful to the Department of Pharmacy, School of Medical Allied

Sciences, Galgotia’s University, Greater Noida, India for providing library facility during

literature survey.

Conflict of interest

Authors have no conflict of interest.

REFERENCES

1. Malviya, R., P. Srivastava and G.T. Kulkarni, Application of mucilages in drug delivery-

A review, Advances in Biological Research, 2011; 5: 01-07.

2. Roohullah, Iqwal Zafar, Fazli Nasir, Muhammad Akhlaq, Sajid Khan Sadozai, Ameer

Zada and Amjad Khan, Sustained Release Carbamezapine Matrix Tablets Prepared by

Solvent-Evaporation Technique Using Different Polymers, Middle-East Journal of

Scientific Research, 2013; 15: 1368-1374.

3. Malviya, R., P. Srivastava, K. Upendra, C.S. Bhargava and P.K. Sharma, Formulation and

comparison of suspending properties of different natural polymers using paracetamol

suspension, IJDDR, 2010; 2: 886-891.

4. Sahoo, S., R. Sahoo and Nayak L. Padma, Tamarind Seed Polysachharide. A versatile

Biopolymer for Mucoadhesive Applications, JPBMS, 2011; 8: 1-12.


2219


5. Malviya, R., P. Srivastava, M. Bansal and P.K. Sharma, Mango Peel Pectin as

Superdisintegrating Agents, Journal of Scientific and Industrial Research, 2010; 69:

688-690.

6. Malviya, R., Extraction and characterization of selected mucilage as a pharmaceutical

excipients, Polim. Med., 2011; 3: 39-44.

7. Dekker J, Rossen J, Bu¨ ller H, Einerhand A. The MUC family: an obituary. Trends

Biochem Sci., 2002; 27(3): 126– 31.

8. Bell S, Xu G, Khatri I, Wang R, Rahman S, Forstner J. N-linked oligosaccharides play a

role in disulphide-dependent dimerization of intestinal mucin Muc2. Biochem J., 2003;

373(Pt3): 893– 900.

9. Perez-Vilar J, Hill R. The structure and assembly of secreted mucins. J Biol Chem, 1999;

274(45): 31751–4. Review article which contains numerous sequence alignments between

the various homologous regions of the different mucin genes.

10. Turner B, Bhaskar K, Hadzopoulou-Cladaras M, LaMont J. Cysteine-rich regions of pig

gastric mucin contain von Willebrand factor and cystine knot domains at the carboxyl

terminal. Biochim Biophys Acta., 1999; 1447(1): 77–92.

11. P.J. Sinko, Martin's physical pharmacy and pharmaceuticals sciences, Micromeritics,

Chapter 19, 5th ed, Lippincott, William &Wilkins, New York, 2005; 553–560.

12. Sheehan J, Kirkham S, Howard M,Woodman P, Kutay S, Brazeau C, et al. Identification

of molecular intermediates in the assembly pathway of the MUC5AC mucin. J Biol

Chem, 2004; 279(15): 15698–705.

13. R.O. Williams III, M. Sriwongjanya, M.K. Barron, Compaction properties of

microcrystalline cellulose using tableting indices, Drug Development and Industrial

Pharmacy, 1997; 23: 695–704.

14. Bell S, Xu G, Forstner J. Role of the cystine-knot motif at the C-terminus of rat mucin

protein MUC 2 in dimer formation and secretion. Biochem J., 2001; 357(Pt1): 203– 9.

15. Lala, P.K., 1981. Practical Pharmacognosy. Calcutta. Lina Guha, 2009; 135.

16. Malviya, R. and G.T. Kulkarni, Extraction and Gum Arabic Coacervates as Excipient in

Fast Characterization of Mango peel pectin as a Disintegrating/ Dissolving Drug Delivery

system. Pharmaceutical excipient, Polim. Med., 2012; 42: 185-190.

17. Malviya, R., P. Srivastava, M. Bansal and P.K. Sharma, Preparation and Evaluation of

Disintegrating Properties of Cucurbita maxima Pulp Powder. International Journal of

Pharmaceutical Sciences, 2010; 2: 395-399.


2220


18. Malviya, R., P. Shukla and Srivastava P. Preparation, Characterization and Evaluation of

Chitosan- FABAD Journal of Pharmaceutical Sciences, 34: 213-223.

19. K. Obae, H. Iijima, K. Imada, Morphological effect of microcrystalline cellulose particles

on tablet tensile strength, International Journal of Pharmaceutics, 1999; 182: 155–164.

Documents

EXTRACTION AND CHARACTERIZATION OF XYLOGLUCAN …