International Journal of Biological Macromolecules 65 (2014) 167175

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Isolation, purication and characterization of galactomannans

as an excipient from Senna tora seeds
Harshal A. Pawar a, , K.G. Lalitha b,1
a

Research Scholar, Ultra College of Pharmacy, 4/235, College Road, Thasildar Nagar, Madurai 625020, Tamil Nadu, India
Professor and Head, Department of Pharmaceutical Chemistry, Ultra College of Pharmacy, 4/235, College Road, Thasildar Nagar, Madurai 625020, Tamil
Nadu, India
b

a r t i c l e

i n f o

Article history:
Received 7 November 2013
Received in revised form 11 January 2014
Accepted 11 January 2014
Available online 20 January 2014
Keywords:
Senna tora
Galactomannan
Seed gum

a b s t r a c t
Seed galactomannans are neutral, heterogeneous polysaccharides widely distributed in nature. The Mannose/Galactose ratios differ from gum to gum, resulting in a change in structure, which in turn, determines
the various industrial applications of seed galactomannans. Senna tora (Family: Fabaceae) is a fast growing and spreading under shrub of which seeds, pods and leaves are extensively used for medicinal
applications. The seeds have been found to be an alternative source of commercial gums. The present
investigation deals with isolation, purication and characterization of galactomannans from the seeds of
Senna tora (S. tora). The galactomannan extraction was based on mechanical separation of the endosperm,
water dissolution, centrifugation and precipitation with acetone. The polysaccharide obtained from S.
tora seeds was characterized by using physicochemical and chromatographic procedures, as well as FTIR,
Mass, 13 C NMR and 1 H NMR spectroscopy. The results indicated that the gum has the basic structure of
galactomannans with a main chain of (14)-linked -d-mannopyranosyl units to which single -(16)d-linked galactopyranosyl units are attached through block pattern. The rheological studies indicated that
the S. tora gum (1%, w/w) solution possesses pseudoplastic ow. The viscosity and other rheological properties conrmed its suitability as an excipient in the development of sustained release delivery systems.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Galactomannans are neutral polysaccharides obtained from the
endosperm of seeds of some Leguminosae plant and they have
several functions, including reserve of carbohydrates [1]. Galactomannans are polysaccharides built up of a -(14)-d-mannan
backbone with single d-galactose branches linked -(16). Their
mannose/galactose (M/G) ratios differ according to the species
[2]. They are water soluble hydrocolloids which form highly viscous, stable aqueous solutions [3]. The main difference between
galactomannans from different plant sources lies in the galactose content as well in its distribution along the mannopyranosyl
backbone [1,4]. The degree of substitution of galactose differs in
the galactomannans extracted from various plants. The differences
of the degree in substitution greatly affect solution properties,
including water solubility, thickening ability and synergistic interactions. Galactomannans can often be used in different forms for

Corresponding author. Tel.: +91 8097148638.

E-mail addresses: hapkmk@rediffmail.com (H.A. Pawar), kg.lalitha@gmail.com
(K.G. Lalitha).
1
Tel.: +91 9894893301.
0141-8130/$ see front matter 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ijbiomac.2014.01.026

human consumption. Featuring different physicochemical properties, galactomannans are a versatile material used for many
applications: they are excellent stiffeners and stabilizers of emulsions, and the absence of toxicity allows their use in the textile,
pharmaceutical, biomedical, cosmetics and food industries [5].
Most galactomannans used in pharmaceutical technology and cosmetics are usually unpuried gums [6]. When associated with other
polysaccharides such as xanthan gum and kappa-carrageenan,
galactomannans can form gels with new properties [710].
The four major galactomannans of commercial importance
in food and non-food industries are guar gum (GG, Cyamopsis
tetragonolobo, M/G ratio: 2:1), tara gum (TG, Caesalpinia spinosa,
M/G ratio: 3:1), locust bean gum (LBG, Ceratonia siliqua, M/G ratio:
3.5:1) and Fenugreek (Trigonella foenum-graecum L., M/G ratio: 1:1)
[11]. Currently the international trends demand the introduction of
alternative sources of seed gums [12] and it is therefore important
to search for alternative renewable sources for e.g. the production
of edible and biodegradable lms and coating materials. In particular, Latin American sources of galactomannans are not well known,
in spite of the rich biodiversity of the local ora and of the favorable
climate for their production [13].
Senna tora (L.) Roxb. belonging to the family Fabaceae is an
annual under shrub which grows all over the tropical countries

168

H.A. Pawar, K.G. Lalitha / International Journal of Biological Macromolecules 65 (2014) 167175

(throughout India, Pakistan, Bangladesh and west China). It grows

well in wasteland as a rainy season weed. It is also known as
Chakramard in Ayurveda [14,15]. Several studies have been conducted throughout the last decade to investigate chemical and
biological properties of S. tora. Antihepatotoxic naphtha-pyrine glycosides were reported to be isolated from the seeds of S. tora [16].
Antioxidant properties and inhibitory effect of the extract of S. tora
have already been reported [17,18]. A recent study was conducted
by the scientists of the Department of Food Science and Nutrition, Catholic University of Daegu, Korea who concluded that S. tora
supplements can help to improve serum lipid status in type-II diabetic subjects without signicant adverse effect [19]. In view of the
easy availability of the plant, the medicinal value of the seeds and
the high demand of seed gums throughout the world, the properties of the seed polysaccharide (gum) obtained from S. tora were
investigated. The present investigation was aimed at isolation and
characterization of puried polysaccharide from the seeds of S. tora
in order to survey its potential applications as an additive/excipient
to neutraceuticals and pharmaceuticals.

2. Materials and methods

2.1. Collection of plant material
The pods of S. tora were collected from Kalyan taluka (District
Thane, Maharashtra) in the month of SeptemberOctober 2010.
The seeds were manually separated; shade dried and kept in a cool,
dry place until further use. Plant was authenticated by Dr. Rajendra D. Shinde, Associate Professor, Blatter Herbarium; St. Xaviers
College, Mumbai and was identied as S. tora (L.) Roxb (Herbarium
Specimen no. 8361). The herbarium specimen of S. tora was stored
in Ultra College of Pharmacy, Madurai for future reference.

2.4. Characterization of gum

2.4.1. Organoleptic evaluation
The Organoleptic evaluation of the puried gum sample was
carried out. The Organoleptic evaluation refers to the evaluation of
color, odor, shape, taste and special features which include touch
and texture. The majority of information on the identity, purity and
quality of the material can be drawn from these observations.
2.4.2. Identication test for gum
The identication of the isolated polysaccharide was carried out
by using the following tests [20,21]:
a. The powder was mounted on a slide with ruthenium red solution
and covered with a cover slip. After a few seconds, it was irrigated
with lead acetate and the excess stain was sucked off with a
blotting paper (lead acetate solution was added to prevent undue
swelling of the test solution). The color of the particles was noted.
b. The powder sample was mounted on a slide with freshly prepared corallin soda solution and covered with a cover slip. After a
few seconds it was irrigated with 25% sodium carbonate solution.
The color of the particles was noted.
c. Polysaccharide was heated with distilled water for some time
and then cooled. Formation of gelatinous mass was noted.
d. To 2 mL of polysaccharide solution, 23 drops of N/50 iodine
solution was added and the color of the particle was noted.
e. Gum solution (35 mL) was treated with 4% borax solution.
2.4.3. Physicochemical evaluation
2.4.3.1. Solubility. The solubility is expressed in terms of parts
representing the number of mililiters (mL) of the solvent in which
1 g of the solid is soluble. Solubility of powder was determined
in different solvents at room temperature as per Indian Pharmacopoeia 1996 [22].

2.2. Isolation of gum

The dried seeds were dehusked and de-germed by mechanical
treatment followed by milling and screening of the endosperm. The
powder was soaked in benzeneethanol solution (1:1) overnight to
remove lipids and then it was dried in vacuum oven. The endosperm
powder of S. tora seeds (10 g) was soaked in 200 mL of distilled
water and stirred under overhead stirrer for 34 h. The viscous solution obtained was passed through the muslin. The marc obtained
was pressed to remove the mucilage and boiled with 200 mL of
water for 1 h. Viscous solution obtained was ltered through muslin
cloth. The marc obtained was not discarded but it was sent for multiple extractions with decreasing quantity of extracting solvent, i.e.,
water with the increase of number of extractions. The isolation was
continued until the material becomes free from mucilage. All the
viscous solutions obtained were mixed together. An equal quantity
of 10% trichloroacetic acid was added to the mixture to precipitate protein. The solution was centrifuged and the supernatant was
precipitated out by addition of acetone in the ratio 1:0.5 with continuous stirring. The coagulated mucilage, which formed as a white
mass was transferred to an evaporating dish and dried in vacuum
oven at 40 C, powdered and stored in airtight containers.
2.3. Purication of gum
The above obtained crude gum was dissolved in warm water,
re-precipitated using ethanol (1:1), dried at 40 C, powdered and
stored in airtight container at room temperature. The process of dissolution in water and precipitation with alcohol was repeated until
an almost white precipitate was obtained. The dried polysaccharide
was milled and sifted with a 60 mesh for further use.

2.4.3.2. pH. The pH of the mucilage was determined using a digital

pH meter. This was done by shaking a 1% (w/v) dispersion of the
gum sample in water for 5 min and the pH determined using a pH
meter. The pH meter was set to neutral (7.4) at a room temperature
and the electrode was immersed into the dispersion. The reading
on the meter was recorded. Triplicate measurements were made
[23].
2.4.3.3. LOD. The inherent moisture in additives may inuence the
stability of dosage form containing moisture sensitive drugs, so the
moisture content of the isolated gum sample was detected by loss
on drying method. The gum sample (1 g) was heated at 105 C until
constant weight in a hot air oven and percentage loss of moisture
on drying was calculated using the formula:
LOD (%) =

weight of water in sample

100
weight of dry sample

2.4.3.4. Ash content. Ash values such as total ash, acid insoluble
ash and water-soluble ash were determined according to Indian
Pharmacopoeia [22,24]. The following procedures were used for
determination of ash values.
2.4.3.4.1. Total ash. About 3 g of sample was accurately
weighed and taken in a silica crucible, which was previously ignited
and weighed. The powder was spread as a ne, even layer on the
bottom of the crucible. The crucible was incinerated gradually by
increasing temperature to make it dull red hot until free from carbon. The crucible was cooled and weighed. The procedure was
repeated to get constant weight. The percentage of total ash was
calculated with reference to air dried sample.

H.A. Pawar, K.G. Lalitha / International Journal of Biological Macromolecules 65 (2014) 167175

2.4.3.4.2. Acid insoluble ash. The ash obtained as described

above was boiled with 25 mL of 2 N HCl for 5 min. The insoluble ash
was collected on an ash less lter paper and washed with hot water.
The insoluble ash was transferred into a silica crucible, ignited and
weighed. The procedure was repeated to get a constant weight. The
percentage of acid insoluble ash was calculated with reference to
the air-dried sample.
2.4.3.4.3. Water-soluble ash. The ash obtained as described for
the determination of total ash was boiled for 5 min with 25 mL of
water. The insoluble matter was collected on ash less lter paper
and washed with hot water. The insoluble ash was then transferred
into silica crucible, ignited for 15 min, and weighed. The procedure
was repeated to get a constant weight. The weight of insoluble matter was subtracted from the weight of the total ash. The difference
of weight was considered as water-soluble ash. The percentage of
water-soluble ash was calculated with reference to the air dried
sample.
2.4.3.5. Specic gravity. Specic gravity of the gum isolates was
determined following the procedure of Skoog and West (1963)
using standardized pycnometer [25]. A dried and clean pycnometer
was lled with 1% gum solution that was previously equilibrated
at 20 C to the indicated level, capped and placed in 25 C constant temperature bath for 30 min. Afterwards, the pycnometer was
dried and weighed. Specic gravity of the solution was obtained by
dividing the weight of the gum solution by the weight of water at
25 C determined previously.
2.4.3.6. Swelling index. The swelling index is the volume (in
milliliters) taken up by the swelling of 1 g of test material under
specied conditions. The swelling indices of the selected hydrogels
were determined by the method described by WHO [26].
Accurately weighed quantity of the gum (1 g), previously
reduced to the required neness, was introduced into a 25 mL glassstoppered measuring cylinder. Twenty-ve milliliters of water was
added and mixture was shaken thoroughly every 10 min for 1 h. It
was then allowed to stand for 3 h at room temperature. Then the
volume occupied by the gum, including any sticky mucilaginous
portion was measured. The same procedure was repeated thrice
and the mean value was calculated.
Swelling index (SI) was expressed as a percentage and calculated
according to the following equation
Swelling index (SI) = 100

X X 
t
0
X0

where X0 is the initial height of the powder in graduated cylinder

and Xt denotes the height occupied by swollen gum after 3 h.
2.4.3.7. Viscosity. Viscosity measurements were carried out with
Ostwalds viscometer tubes after 24 h of hydration of the gum. For
preparing the gum solutions (0.05%, w/v), 50 mg of the gum was dissolved in the minimum quantity of water and then made volume
up to 100 mL with water. The prepared solution was agitated vigorously for approximately 15 min until the solution become viscous
and homogeneous.
2.4.4. Differential scanning calorimeter (DSC)
Thermal properties of S. tora gum powder were studied using
Differential Scanning Calorimeter.
Nitrogen, at the rate of 50 mL/min, was used as purge gas; 2 mg
of powdered material was sealed in aluminium pan and heated
from 30 C up to 500 C at the rate of 10 C/min, followed by a
cooling cycle back to 30 C at the same rate.

169

2.4.5. X-ray diffraction

Diffraction pattern of powdered S. tora polysaccharide was
recorded with an X-ray diffractometer. X-ray diffraction was performed at room temperature (30 C) with a diffractometer; target,
lter, Ni; voltage, 40 kV; current 30 mA; time conCu ( = 1.54 A),
stant 10 mm/s; scanning rate 2 /min; measured from 5 to 50 .
2.4.6. Scanning electron microscope (SEM)
To study the surface characteristics of gum, SEM of the powder
was taken. The SEM photomicrographs of native and swelled gum
were recorded by using a scanning electron microscope (JEOL 5400,
Japan).
2.4.7. Gel permeation chromatography (GPC)
The S. tora gum solution (1 mL, 0.5% in water) was loaded
onto Sepharose CL-4B column (bed volume 180 mL) pre-calibrated
with T-series dextran standards of known molecular weight (Mw)
and eluted with water at a ow rate of 18 mL h1 . The molecular
weight of polysaccharide was determined from the standard graph
obtained by plotting the log Mw of the standards versus Ve/Vo (Ve
elution volume of the standards and Vo void volume).
2.4.8. Estimation of total sugar content
The total sugar content was determined according to the procedure of Dubois et al. [27]. About 10 mg of S. tora gum was dissolved
in 100 mL distilled water. One milliliter of 5% phenol solution
was added to 1 mL gum solution in acid-washed test tube. Five
milliliters concentrated H2 SO4 was directly and rapidly added to
the test tubes. The solution was allowed to stand for 10 min and
was shaken. Absorbance of the solution was read at 490 nm using
a UVvisible spectrophotometer.
Standard calibration curve was prepared using solutions ranging
from 50 to 100 mcg/mL. Solutions were prepared in triplicates from
1000 mcg/mL stock glucose solution.
2.4.9. Identication of gum components by TLC
Boiled 200 mg of the gum sample in 20 mL of 10% sulfuric acid
for 3 h. The solution was cooled and added excess of barium carbonate, mixing with a magnetic stirrer until the pH of the solution
became 7. Filtered the solution and evaporated the ltrate in a rotatory evaporator at 3050 C under vacuum until a syrupy residue
was obtained (Gum hydrolysate). The obtained residue was dissolved in 10 mL of 40% methanol and TLC was performed using
below mentioned procedure.
TLC of the gum hydrolysate was performed on precoated silica
gel GF 254 plates (Stationary Phase) using mixture of n-butanol,
isopropyl alcohol and water in the ratio 11:6:3 as solvent system
(Mobile Phase). Galactose and mannose were used as standard.
Detection was done by spraying the plate with 10% H2 SO4 solution in methanol and the plate was kept in oven at 110 C for 1 h.
The Rf values for the separated spots were calculated and compared
with Rf value of pure galactose and mannose and values reported
in the literature [28,29].
2.4.10. Determination of mannose/galactose ratio (M/G ratio)
Mannose and galactose content in the polysaccharide were
determined in triplicate using Cleggs method (1958) as described
below:
Preparation of dilute galactose and mannose solution:
100 mg of galactose was dissolved in 100 mL distilled water.
10 mL of this solution was diluted to 100 mL with distilled water.
The same procedure was repeated to make the dilute mannose
solution.
Preparation of sample solution:
10 mg of S. tora gum was dissolved in 100 mL distilled water.
Preparation of Anthrone reagent (0.1%):

170

H.A. Pawar, K.G. Lalitha / International Journal of Biological Macromolecules 65 (2014) 167175

Table 1
Organoleptic features of the puried S. tora seed gum.
Color

Odor

Taste

Shape

Touch and texture

Dull brown

Odorless

Tasteless

Irregular

Hard and rough

100 mg Anthrone was dissolved in 100 mL sulphuric acid

(270 mL concentrated H2SO4 was dissolved in 300 mL distilled
water).
Procedure:
One milliliter from each dilute solution of sample, galactose and
mannose were pipetted into a series of test tubes 1, 2, and 3, respectively. Five milliliter of the Anthrone reagent was added to each test
tube. The solutions were allowed to cool for 12 min. Absorbance of
each solution was read at 360 nm using a UVvisible spectrophotometer. The galactose and mannose contents were found out using
following formula and M/G ratio was determined.
Galactose or mannose content (%) =

25 B
SA

where, B is the reading of sample, A is the reading of dilute galactose

(or mannose) and S is the weight of origin sample.
2.4.11. Investigation of the structure of the polysaccharide
No one method of determining structure will give answers to all
the questions for determination of the structure of polysaccharides.
In addition, each method has limitations so it is always desirable
to obtain information on particular aspects of structure using more
than one method.
An IR spectrum of the polysaccharide was taken using a
Fourier transform infrared spectrometer (FTIR, SHIMADZU). The
polysaccharide (S. tora gum) was ground with spectroscopic grade
potassium bromide (KBr) powder and then pressed into 1 mm pellets for FTIR measurement in the wavenumber range of 400 and
4000 cm1 .
Mass spectrum of the puried gum sample was taken by using
electrospray ionization (ESI) technique with Waters Quadrapole
Mass Spectrometer (Switzerland). ESI-MS/MS was performed using
API 4000 LC/MS/MS (Applied Biosystem). The gum sample was dissolved in solvent mixture of dimethylsulfoxide and water (1:1).
Nuclear magnetic resonance (NMR) spectra were obtained using
a Bruker Avance 300 MHz NMR spectrometer (Bruker Co., Billerica,
MA). The sample (Gum hydrolysate) solutions were prepared in
D2 O for 1 H NMR and 13 C NMR. 1 H chemical shifts were referenced
to internal D2 O.
2.4.12. Rheological study of S. tora gum
Rheological properties are much useful in behavior and predictive information for various pharmaceutical products, as well as to
get the knowledge of the effects of processing, formulation changes
and aging phenomena. In particular to the gum materials, viscosity
is the main parameter to predict the quality of the material and
rheological behavior is a useful tool for its applicability in various
Pharmaceutical dosage forms as an excipient.
The rheological behavior of 1% (w/v) solution of S. tora gum was
studied at different RPM using Brookeld viscometer (Spindle no.
5, time: 5 min, 100 mL beaker).

Table 2
Physicochemical evaluation of S. tora seed gum.
Parameter

Results

Solubility

Soluble in hot water forming viscous

colloidal solution but insoluble in
methanol, ethanol, acetone, DMSO and
ether
6.57.3 (near to neutral, less irritating
and hence suitable for uncoated
tablets)
6.7% (w/w)
Total 1.2% (w/w)
Acid insoluble 0.26% (w/w)
Water soluble 0.34% (w/w)
0.9929

pH

Loss on drying
Ash content

Specic gravity (1%, w/v,

solution)
Swelling index
Viscosity (0.05%, w/v, in water)

84%
1.714 cP

heated with distilled water. All these tests indicated that the isolated polysaccharide is gum. In the iodine test, the particles did
not stain blue, indicating the absence of starch. With borate ions,
gum solution gave rise to cohesive gels indicating the presence of
galactomannans in gum.
The results of physicochemical evaluation are summarized in
Table 2. The pH values of 1% solution of the S. tora gum was found
to be slightly acidic or near neutral, which indicated that the gum
is non-irritating to the mucous membrane of buccal cavity and gastrointestinal tract, and could be used for the development of buccal
and oral drug delivery systems.
Differential scanning calorimetry (DSC) measures the heat loss
or gain, resulting from physical or chemical changes within a
sample as a function of temperature. A sharp symmetric melting
endotherm can indicate relative purity, whereas broad asymmetric
curve suggests impurities or more than one thermal process. DSC,
because of its sensitivity and accuracy, has been extensively used
to study the phase transitions of polymers. The DSC thermogram of
S. tora seed polysaccharide is shown in Fig. 1.
S. tora gum shows distinct feature in DSC having one endotherm
and other at relatively higher temperature due to decomposition of
main chain. The seed polysaccharide exhibited broad endothermic
peak at about 80 C (Glass transition temperature, Tg ) due to loss
of free/bound water present in the S. tora seed polysaccharide. The

3. Results and discussion

The gum obtained from S. tora seeds was an amorphous free
owing powder with dull brown color (yield = 35%w/w). The results
of the Organoleptic evaluation are summarized in Table 1.
With ruthenium red and corallin soda, the particles did not stain
pink and a gelatinous mass was not formed when the powder was

Fig. 1. DSC thermogram of S. tora seed polysaccharide.

H.A. Pawar, K.G. Lalitha / International Journal of Biological Macromolecules 65 (2014) 167175

3000

0.9

2000

0.8
0.7

20

40

60

2
Fig. 2. X-ray diffraction pattern of S. tora seed polysaccharide.

Absorbance

1000
0

171

0.6
0.5

y = 0.0083x
R = 0.9949

0.4
0.3
0.2
0.1
0

20

40

60

80

100

120

Concentraon in g/mL
Fig. 5. Calibration curve of glucose for Phenol Sulphuric Acid Assay.

Fig. 3. SEM of native S. tora seed polysaccharide.

latter step is followed by depolymerization which proceeds due

to cleavage of glycosidic linkage. The small endothermic peak at
325.83 C is due to melting/decomposition of polysaccharide. The
broad nature of S. tora gums thermogram shows that it is amorphous in nature even though it looks crystalline to unaided eyes.
The X-ray diffraction pattern (Fig. 2) of S. tora seed gum did not
show any characteristic peak, which indicates that the obtained
polysaccharide is completely amorphous.
To study the surface characteristics of gum, SEM of the powder was taken. The scanning electron micrographs of S. tora gum
powder (Native and Swelled) are shown in Figs. 3 and 4. SEM of
native gum shows distinct particles of fairly dened shape. The
micrograph of S. tora gum at 50 magnication (Fig. 3) shows some
irregular-shaped particles which points to the amorphous nature
of the powder. The SEM of S. tora gum exhibited rough surface with
pores and crevices on it (Fig. 3). Thus this micrograph further conrms the results from DSC. If the surface is rough, drug release will
be retarded because of the entrapment of drug particles in the pores
and crevices. Hence, it can be concluded that S. tora gum can sustain the drug release because of its rough surface. From the SEM,
it was also evident that the particle size of the powder was not

Fig. 4. SEM of swelled S. tora seed polysaccharide.

uniform and the size distribution was not within a narrow range.
The powder contains larger to ultra-ne particles. This might be the
reason for the heavy nature of the powder. The molecular weight
of puried S. tora gum was found to be 198 kDa by gel permeation
chromatography.
Fig. 5 represents the standard curve of glucose used in the Phenol Sulphuric Acid Assay to determine the total sugar content of
the gum isolate. Using the proposed method, the calibration curve
was found to be linear in the range of 50100 g/mL. A correlation coefcient of 0.9949 indicates good linearity between the
concentration and absorbance. The % relative standard deviation
(% RSD) of 0.40 indicates that the used method is precise and
accurate. The total polysaccharide content of S. tora gum was calculated using regression equation obtained from the calibration
curve. Total sugar content in S. tora gum was found to be 92.06%
(w/w) (mean of three determinations).
TLC of the hydrolysate showed two spots. The spots were identied as galactose and mannose by comparing their Rf values with
standards of pure galactose and mannose. Galactose was liberated
rst followed by mannose. The average mannose to galactose ratio
(M/G ratio) was found to be 5.1.
Fig. 6 represents FTIR spectrum of S. tora polysaccharide. The
IR spectra of polysaccharide shows peaks at 813 and 875 cm1
that are related with the presence of anomeric congurations
( and conformers) and glycosidic linkages, attributed to -dgalactopyranose units and -B-mannopyranose units, respectively
[31,32]. The broad band between 1198 and 983 cm1 results
from the stretching vibration of C O in C O H bonds (e.g. glycosidic bonds) and is related with the galactomannans sugar
composition. The peak at 1149 cm1 corresponds to bending
vibrational modes of C O, present in the pyranose ring, while
the broad band between 1134 and 983 cm1 is a characteristic

Fig. 6. FTIR spectrum of the S. tora polysaccharide.

172

H.A. Pawar, K.G. Lalitha / International Journal of Biological Macromolecules 65 (2014) 167175

Fig. 7. ESI-MS spectra of S. tora gum.

contribution of C OH bending [33]. The broad bands ranging

between 28003000 and 31003500 cm1 are attributed to C H
stretching and to O H stretching vibration, respectively [34]. In
the anomeric region (950700 cm1 ), gum exhibited the obvious
characteristic absorption at 813 cm1 , suggesting the existence of
mannose [35,36].
The analysis of ESI-MS allows studying intact oligosaccharides,
even when present in mixtures and with low abundances without
any manipulation/derivatization being required [37].
An ESI-MS spectrum of gum shows characteristic fragments at
m/z: 591.4, 527.5, 506.5, 490.5, 467.5, 428.5, 420.3, 392.5, 371.5,
344.3, 313.2, 265.4, 217.3, 195.3, 175.4 and 145.4.
Fig. 7 shows the ion at m/z 527.5 (loss of 60 Da), m/z 467.5 (loss
of 90 Da) and, with higher abundance, the ion at m/z 428.5 (loss of
120 Da) corresponding to a 16 type linkage. This fragmentation
pathway allowed to identify polysaccharide as being composed of
mannose units linked by a (14) linkage and an additional galactose
residue (16) linked. The ion at m/z 344.2 (C12 H24 O11 ) corresponds
to two mannose units linked through (14) linkage [5].

Fig. 8. Fragmentation pattern (cleavage of two bonds within the sugar ring) of sugar.

Fig. 9. ESI-MSMS spectra of S. tora gum.

H.A. Pawar, K.G. Lalitha / International Journal of Biological Macromolecules 65 (2014) 167175

173

Table 3
Chemical shifts (in ppm) and the corresponding carbon atoms for 1 H NMR signals
of S. tora gum.

Fig. 10.

13

C NMR spectra of galactomannan from the Senna tora seeds, in D2 O.

In order to conrm the proposed oligosaccharide structures

observed in the ESI-MS spectra, the sample was further analyzed by
LCESI-MS/MS. Oligosaccharide cleavages under ESI-MS/MS conditions are the result of glycosidic cleavages between two sugar
residues and of cross ring cleavages (cleavage of two bonds within
the sugar ring as shown in Fig. 8).
LCMSMS spectrum (Fig. 9) of gum shows characteristic fragment at: 514.9, 485.3 (loss of 3 hexose units 2H2 O), 448.9, 393
(loss of 3 H2 O), 378.9 (two hexose units + H2 O), 325.2 (two hexose units 2H2 O), 311.0, 248.9, 232.9, 180.9 (hexose unit), 175.0,
138.9, 113 (hexose unit 2H2 O-HCHO), 102.9 (loss of water without cyclization, 103 Da), 96.9, 91.0 (pyranose), 81.0 and 62.0.
The 13 C NMR spectrum of galactomannan is shown in Fig. 10.
Well resolved signals correspond to -d-mannopyranose and d-galactopyranose residue were observed. The 1 H NMR spectrum

Fig. 11.

H NMR spectra of galactomannan from the Senna tora seeds, in D2 O.

Chemical shift (ppm)

Corresponding C-atom (moiety)

4.899

H-1 () {-d-sugar moieties}

4.683

H-2 () {-d-sugar moieties}

3.999
3.854
3.621

H2 to H6

of the galactomannan is shown in Fig. 11. In the anomeric region

two distinct signals corresponds to -d-mannopyranose and d-galactopyranose were observed. The high resolution 13 C NMR
spectra of isolated polysaccharide contained two main signals
in the anomeric region at 101.379 ppm and 99.932 ppm, which
were assigned to the -d-mannopyranosyl and -galactopyranosyl
residues, respectively [3840]. The ratio of galactose and mannose
can also be obtained directly from the relative areas of the signals
for H1 for both of them, and the ratio was 1:5.17 which conrms
the previous obtained results. The chemical shifts of galatomannan
summarized in Tables 35 are in accordance with those reported
in the literature [41].
The above results obtained indicated that the polysaccharide
isolated from seeds of S. tora was a galactomannan with a chain
of ve d-mannopyranosyl residue linked through (14) linkage
and a -d-galactopyranosyl residue at O-6 of mannose unit. The
proposed structure is shown in Fig. 12.
The rheological property of the gum solution was studied by
plotting the rheogram. Fig. 13(Graph-I) represents rheogram of
shear stress versus shear rate. It was found that the curve obtained
from measurement at decreasing shear rate was super imposed
on that obtained from measurements taken at increasing shear
rate. Fig. 14(Graph-II) represents rheogram of viscosity versus shear
rate. As the rate of shear increases, the viscosity of gum solution
decreases. This indicates that 1% (w/v) solution of S. tora gum exhibited pseudoplastic ow and the system was shear thinning.
Table 4
Chemical shifts (in ppm) and the corresponding carbon atoms for 13 C NMR signals
of S. tora gum.
Chemical shift (ppm)

Corresponding C-atom (moiety)

Intensity

101.379

C1()

0.74

99.932

C1()

0.44

73.0303
71.2276
71.1204
70.4660
70.0551
69.8123
68.7786
66.3245

C-2 to C-5

0.75
0.55
0.81
0.88
0.42
0.44
0.40
0.79

61.8052
61.4983

CH2 OH

0.45
0.86

56.0115
55.5820
55.2552

O-CH3

4.76
0.40
0.65

Fig. 12. Structure of S. tora seed polysaccharide.

174

H.A. Pawar, K.G. Lalitha / International Journal of Biological Macromolecules 65 (2014) 167175

Table 5
Assignment of 13 C NMR signals of S. tora gum in D2 O.
Units

C-1

-d-Galactopyranosyl
-d-Mannopyranosyl

99.9316
101.3790

C-2
68.7787
70.4660

90
80

Shear Stress

70
60
50
40
30
20
10
0

20

40

60

80

100

120

Shear Rate

C-3

C-4

C-5

C-6

70.0551
71.1204

69.8123
73.0303

71.2276
67.3245

61.4983
61.8052

application do not cause pollution or disturb the ecosystem. Galactomannans are used in various pharmaceutical dosage forms such
as tablets or capsules, hydrogels and lms as an excipient. Besides
the simple use as inert excipient, these polysaccharides play role
in the modication of drug release, especially in colonic environmental, as a matrix or coating material. It has been concluded from
the above study that the polysaccharide (gum) isolated from S. tora
seeds contains galactomannans. The viscosity and other rheological
properties conrmed its suitability as an excipient in the development of sustained release delivery systems. Thus there is need to
investigate further S. tora polysaccharide as an excipient in different pharmaceutical dosage forms. It may provide an alternative to
synthetic or semisynthetic excipients/polymers currently used in
the pharmaceutical industry.

Fig. 13. Rheogram of shear stress versus shear rate (Graph-I).

References

12000

Viscosity in Cps

10000
8000
6000
4000
2000
0

20

40

60
Shear Rate

80

100

120

Fig. 14. Rheogram of viscosity versus shear rate (Graph-II).

Viscosity is the main parameter to assess the quality of natural

gums. The applications of any natural gum are dependent on its
viscosity and other rheological properties. For any polymer to be
used in slow release hydrophilic matrix systems, it should possess
certain characteristics like fast hydration of the polymer, high gel
strength and should be stable during the shelf life of the product.
The results indicate that the polysaccharide isolated from S. tora
seeds possess pseudoplastic ow and good swelling capacity. S. tora
seed polysaccharide hydrates quickly and swells rapidly and forms
a thick viscous gel around it. This is the most important criterion
required for hydrophilic matrix tablets. The viscosity and other rheological properties conrmed its suitability in the development of
sustained release delivery systems.
4. Conclusion
The pharmaceutical applications of galactomannans obtained
from different commercial and noncommercial sources have been
extensively studied over the past decade. Galactomannans show
potential in the global trend toward the use of more plantbased products for ecological motives, and their production and

[1] J.S.G. Reid, M.E. Edwards, in: A.M. Stephen (Ed.), Food Polysaccharides and Their
Application, Marcel Dekker Inc., New York, 1995, pp. 155186.
[2] M.S. Kok, S.E. Hill, J.R. Mitchell, Food Hydrocolloids 13 (1999) 535542.
[3] H. Neukom, Galactomannans: Properties and Applications, 22, Lebensmmitel
Wissenschaft und Technologie, London, 1989, pp. 4145.
[4] I.C.M. Dea, A. Morrison, Advances in Carbohydrate Chemistry and Biochemistry
31 (1975) 241.
[5] C.A. Migue, W.S. Bartolomeu, S. Joana, et al., Carbohydrate Polymers 83 (2011)
179185.
[6] M. Uner, T. Altnkurt, IL Farmaco 59 (2004) 567573.
[7] C.T. Andrade, E.G. Azero, L. Luciano, et al., International Journal of Biological
Macromolecules 27 (2000) 349353.
[8] E.G. Azero, C.T. Andrade, Journal of the Brazilian Chemical Society 17 (2006)
844850.
[9] T.M.B. Bresolin, M. Milas, M. Rinaudo, et al., International Journal of Biological
Macromolecules 26 (1999) 225231.
[10] M.J. Hernandez, J. Dolz, M. Dolz, et al., Food Science and Technology International 7 (2001) 383391.
[11] V.D. Prajapati, G.K. Jani, N.G. Moradiya, N.P. Randeria, B.J. Nagar, N.N. Naikwadi, B.C. Variya, International Journal of Biological Macromolecules 60 (2013)
8392.
[12] H. Joshi, V.P. Kapoor, Carbohydrate Research 338 (2003) 19071912.
[13] E.G. Azero, C.T. Andrade, Polymer Testing 21 (2002) 551556.
[14] I. Anita, R. Pranjali, L. Abhijeet, et al., International Imperial Journal of Pharmacognosy & Natural Products 2 (2012) 1423.
[15] C.S. Narayan, S. Rangaswami, Current Science 25 (1956) 559.
[16] S.M. Wong, M.M. Wong, O. Selingmann, et al., PlantaMedica 55 (1989)
276280.
[17] C.H. Wu, C.L. Hsieh, J.Y. Song, et al., Journal of Agricultural and Food Chemistry
49 (2001) 25792586.
[18] G.C. Yen, D.Y. Chuang, Journal of Agricultural and Food Chemistry 8 (2000)
27602765.
[19] S.H. Cho, T.H. Kim, N.H. Lee, et al., Journal of Medicinal Food 8 (2005) 311318.
[20] P.K. Lala, Practical Pharmacognosy, Lina Guha, Calcutta, 1981.
[21] F. Smith, R. Montagomery, The Chemistry of Plant Gums and Mucillages, Reinhold Publishing Corporation, New York, 1959.
[22] Indian Pharmacopoeia, vol. 556, Ministry of Health and Family Welfare, Government of India, Controller of Publication, New Delhi, 1996, pp. A100A111.
[23] E.E. Jarald, S. Sumati, S. Edwin, et al., Indian Journal of Pharmaceutical Education
and Research 46 (2012) 211216.
[24] S.S. Rohokale, Y.D. Dhanorkar, V. Pahuja, et al., Journal of Chronotherapy and
Drug Delivery 3 (2012) 4154.
[25] D.A. Skoog, D.M. West, Fundamentals of Analytical Chemistry, 4th ed., HoltRinehart and Winston, Philadelphia, NY, 1963, pp. 880.
[26] World Health Organization, Quality Control Methods for Medicinal Plant Material, WHO, Geneva, 1998, pp. 45.
[27] M. Dubois, C. Gileska, J.K. Hamilton, Analytical Chemistry 28 (1956) 350356.
[28] E.L. Hirst, J.K.N. Jones, Discussions of Faraday Society 7 (1949) 268.
[29] S.A.I. Rizvi, P.C. Gupta, R.K. Kaul, Planta Medica 20 (1971) 2428.
[31] S.D. Figueir, J.C. Ges, R.A. Moreira, et al., Carbohydrate Polymers 56 (2004)
313320.
[32] B.M. Prado, S. Kim, B.F. zen, et al., Journal of Agricultural and Food Chemistry
53 (2005) 28232829.

H.A. Pawar, K.G. Lalitha / International Journal of Biological Macromolecules 65 (2014) 167175
[33] M.R.S. Prashanth, K.S. Parvathy, N.S. Susheelamma, et al., Food Hydrocolloids
20 (2006) 11981205.
[34] S.N. Yuen, S.M. Choi, D.L. Phillips, et al., Food Chemistry 114 (2009) 10911098.
[35] Y. Peng, L. Zhang, F. Zeng, et al., Carbohydrate Polymers 54 (2003) 297303.
[36] L. Zhang, L. Yang, Q. Ding, et al., Carbohydrate Research 270 (1995) 110.
[37] F.M. Nunes, M.R. Domingues, M.A. Coimbra, Carbohydrate Research 340 (2005)
16891698.

175

[38] G.M. Gu-bitza, B. Laussamauer, M. Schubert-Zsilavccz, et al., Enzyme and Microbial Technology 26 (2000) 1521.
[39] C.L.O. Petkowicz, M.R. Sierakowski, M.S. Joana La, et al., Phytochemistry 49
(1998) 737743.
[40] S.E.C. Whitney, J.E. Brigham, A.H. Darke, et al., Carbohydrate Research 307
(1998) 299309.
[41] H.N. Cheng, N.G. Thomas, Polymer Reviews 52 (2012) 81114.