International Journal of Biological Macromolecules 115 (2018) 871–875

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International Journal of Biological Macromolecules

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

Extraction of polysaccharide from fruits of Cordia dichotoma G. Forst

using acid precipitation method and its physicochemical characterization
Harshal Ashok Pawar ⁎, Amit Jagannath Gavasane, Pritam Dinesh Choudhary
Dr. L. H. Hiranandani College of Pharmacy, Ulhasnagar 421003, Maharashtra, India

a r t i c l e i n f o a b s t r a c t

Article history: The objective of the present work was to isolate and characterize polysaccharide from fruits of Cordia dichotoma
Received 12 April 2018 G. Forst. (Family Boraginaceae). Polysaccharide was isolated by using 1% Hydrochloric acid solution. The isolated
Received in revised form 16 April 2018 polysaccharide was tested for physicochemical characteristics such as solubility, pH (1% w/w in water), swelling
Accepted 27 April 2018
capacity, loss on drying, ash value, bulk and tapped density, Carr's capacity, Hausner's ratio and angle of repose.
Available online 30 April 2018
Also isolated polysaccharide was characterized by Differential scanning colorimeter (DSC), Estimation of total
Keywords:
sugar content, Rheological study and infrared spectroscopy (FT-IR). The isolated mucilage showed positive re-
Cordia dichotoma sults for Molisch's test and negative for Ruthenium red test which indicated presence of carbohydrate and
Polysaccharide gum. The result of physicochemical characteristics reveals that isolated Cordia dichotoma polysaccharide pos-
Characterization sesses good flow properties. The total polysaccharide content of Cordia dichotoma polymer isolate was found to
be 86.24% (w/w). From this study it can be concluded that the polysaccharide isolated from Cordia dichotoma
fruits has the required properties and could be used as an excipient for pharmaceutical dosage forms.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction Literature survey revealed that Cordia polysaccharide was isolated

Cordia dichotoma Forst (Cordia dichotoma) a small to moderate size
plant of family Boraginaceae. Cordia dichotoma is one of the traditional
medicinally important deciduous plants available all over India.
The whole plant of Cordia dichotoma is edible and is used as food. Im-
mature fruits are pickled and are also used as vegetable Mixture of
flower and curd applied two times in a day used to protect body against
heavy sun heat waves. Plant parts such as leaves, fruit, bark and seed
have been reported for possessing antidiabetic, antiulcer, anti-
inflammatory, immune-modulator and analgesic activity [1].
Gums and mucilage's are polysaccharide complexes formed from
sugar and uronic units. They are insoluble in alcohol but dissolve or
swell in water. The polysaccharide may be produced naturally from
plants or produced synthetically. Natural gums are obtained as exudates
or extractives from various plants and plant parts [2].
Gums in general may be classified as acidic, neutral or basic. Natural
gums are either acidic or neutral. No basic gum occurs in nature. Exam-
ples of acidic gums are acacia, tragacanth and albizia gums. Examples of
neutral gums are asparagus gum and plantago seed gums. Herbal gums
are being widely used in the process of development of new pharma-
ceutical formulations [3].
⁎ Corresponding author at: Dr. L. H. Hiranandani College of Pharmacy, Smt. CHM
Campus, Opp. Ulhasnagar Railway Station, Ulhasnagar 421003, Maharashtra, India.
E-mail addresses: hapkmk@rediffmail.com, harshal.dlhhcop@gmail.com (H.A. Pawar).
from fruit using solubilizing its pulp into water followed by its precipi-
tation with organic solvent [4]. This process is costly since more amount
of organic solvent in required during isolation. Literature, research arti-
cles and patents reveal the various applications of Cordia dichotoma gum
isolated using acetone in pharmaceuticals like emulsifier, controlled
drug delivery system, microparticulate drug delivery, nanoparticles
etc. The gum (Polysaccharide) obtained from the fruit of Cordia
dichotoma has been reported to be used as binder in tablet formulation.
It was also used in the preparation of mucoadhesive dosage form, fabri-
cation of sustained release matrix tablets, transdermal films, beads and
ophthalmic drug delivery system [5–10].
The objective of the present research work was to isolate the poly-
saccharide from fruits of Cordia dichotoma G. Forst using acid precipita-
tion method and perform its physicochemical characterization in order
to evaluate its suitability as a pharmaceutical excipient.
2. Materials and methods
2.1. Collection of materials
The fruits were collected from the tree Cordia dichotoma G. Forst.
Family Boraginaceae in the month of June 2014 from Mohol, Village of
Solapur District (Maharashtra), India. Plant was authenticated by Dr.
Rajendra. D. Shinde, Associate Professor, Blatter Herbarium; St. Xavier's
College, Mumbai and was identified as Cordia dichotoma G. Forst.

https://doi.org/10.1016/j.ijbiomac.2018.04.146
0141-8130/© 2018 Elsevier B.V. All rights reserved.
872 H.A. Pawar et al. / International Journal of Biological Macromolecules 115 (2018) 871–875

Belonging to family Boraginaceae. The specimen matched with Blatter Table 2

Herbarium specimen number 1702 of S. M. Almeida. Preliminary phytochemical test of Cordia dichotoma gum.

Test Observations Inference

2.2. Isolation and purification of polysaccharide from Cordia dichotoma Molisch's test A violet ring appears at the junction of the Carbohydrate
fruits two liquids. present
Fehling's test Formation of brownish-red precipitate. Reducing sugar
The seeds were separated from fruits and remaining part was mixed present
Iodine test No deep blue colour Starch absent
with water to dissolve the polysaccharide. The mixture was stirred for
Biuret test No violet purple colouration Proteins may be
3 h. This was then passed through muslin cloth to get a viscous solution. absent
The polysaccharide was precipitated by adding equal volume of 1% Hy- Ruthenium red No pink colouration Gum present
drochloric acid solution. The polysaccharide was separated by passing test
through muslin cloth and dried overnight at 40 °C. The precipitated
dried polysaccharide was pulverized using mortar pestle and the pow-
der obtained was passed through 100# sieve [11].
The obtained polysaccharide was purified by giving water washing
so as to make it free from acid. This procedure was repeated 5–6 times. The angle of repose of powder was carried out by the fixed funnel
method. The accurately weighed quantity of powder mix was taken in
2.3. Characterization of polysaccharide a funnel. The height of the funnel was maintained in such a way that
the tip of the funnel just touched the apex of the heap of the powder.
2.3.1. Organoleptic evaluation The powder was allowed to flow through the funnel without any resis-
Organoleptic evaluation of purified and sieved polysaccharide was tance on to the surface. The diameter and height of the powder cone
carried out which include colour, odour, taste, shape and texture. was measured. Angle of repose was determined using the subsequent
equation:
2.3.2. Preliminary phytochemical evaluation
Preliminary phytochemical screening was carried out on obtained h
polysaccharide according to standard procedure reported in literature tanθ ¼
r
[12–15].
where, h and r are the height and radius of the powder cone.
2.3.3. Physicochemical evaluation
D. Bulk density and tapped density
The dried polysaccharide powder was evaluated for its physico-
chemical properties as per standard procedure reported in literature.
A. Solubility Powder weighing 5 g from each formula was introduced into a 25-
mL measuring cylinder. It was initially shaken lightly to break any ag-
glomerates formed. Initial volume was noted and the cylinder was
Solubility of isolated polysaccharide was determined in different sol- allowed to fall under its own weight onto a hard surface from the height
vents as per procedure given in Indian pharmacopoeia 1996 [16]. of 2.5 cm at 2-second intervals. The tapping was continued until a con-
stant volume was observed. LBD and TBD were calculated using the fol-
B. Powder characteristics
lowing formulas:

weight of powder
Powder of isolated Cordia dichotoma fruit polysaccharide was char- Bulk density ¼
volume of packing
acterized for different parameters like angle of repose, bulk density,
tapped density, Carr's capacity and Hausner's ratio [17].
Weight of powder
Tapped density ¼
C. Angle of repose Tapped volume of packing

Table 1
Physicochemical evaluation of isolated Cordia dichotoma polysaccharide.

Parameters Results

Solubility Partially soluble in water, insoluble in acetone,

ethanol, methanol, DMSO and ether
Powder Bulk 0.6385 g/cm3
characteristics density
Tapped 0.7320 g/cm3
density
Angle of 11.44°
repose
Carr's 12.77%
capacity
Hausner's 1.14
ratio
pH (1% w/v) 4.85 to 6
Specific gravity (1% w/v 0.9958 g/mL
solution)
Swelling capacity 200%
Viscosity (0.1% w/v, in 0.86 cP
water)
Loss on drying (LOD) 15.6% w/w
Fig. 1. DSC thermogram of Cordia dichotoma gum.
 H.A. Pawar et al. / International Journal of Biological Macromolecules 115 (2018) 871–875 873

Fig. 4. Rheogram of shear stress versus shear rate.

G. Loss on drying
Fig. 2. Standard curve of glucose.

One-gram polysaccharide sample was heated in hot air oven at

E. Compressibility capacity and Hausner's ratio 105 °C till constant weight was observed. The loss in moisture con-
tent was then calculated by the following equation.

Weight of water in sample

The following formula was used to determine the compressibility ca- %LOD ¼  100
Weight of dry sample
pacity of granules:

ðTapped density−Bulk densityÞ

Carr0 s capacity ¼  100 H. Specific gravity
Tapped density

Hausner's ratio was calculated by the following formula: Specific gravity of the polysaccharide using Skoog and West's
method [20] using a pycnometer [20]. One percent solution of gum
Tapped density was prepared and poured into a dry calibrated pycnometer. It was
Hausner0 s Ratio ¼
Bulk density capped and weighed at room temperature. Similarly weight of pycnom-
eter with water was noted at room temperature.
I. Swelling Capacity
F. pH

Swelling capacity of isolated polysaccharide was determined. One

pH of 1% (w/v) gum dispersion in water was checked after stirring gram finely grounded gum was introduced into 25 mL measuring cylin-
for 5 min using a digital pH meter whose electrode was set neutral be- der. This was tapped 200 times and initial volume was noted. Water was
fore immersing the electrode into the dispersion. Triplicate measure- added up to 24 mL. The cylinder was shaken intermittently every
ments were made [18,19]. 10 min for 1 h. It was then allowed stand for 24 h at room temperature.

Fig. 3. FTIR spectra Cordia dichotoma polysaccharide.

874 H.A. Pawar et al. / International Journal of Biological Macromolecules 115 (2018) 871–875
Fig. 5. Rheogram of viscosity versus shear rate.
The volume occupied by the gum, including any sticky mucilaginous
portion was noted as final volume. Swelling capacity was calculated as
per following equation [21].
Final volume
Swelling capacity ¼  100
Initial volume
J. Viscosity
Ostwald's viscometer was used for determining the viscosity of poly-
saccharide solution (0.1% w/v). 0.1 g gum was kept for hydration in
25 mL water for 24 h. This gum solution was then made up to 100 mL
with water and homogenized using high speed homogenizer at
10,000 revolutions per minute (rpm) for 10 min. Viscosity of this solu-
tion was determined.
2.3.4. Differential scanning colorimeter (DSC)
DSC was performed using a 10 mg sample from 30 °C to 305 °C at a
heating rate of 10 °C/min in a synthetic air atmosphere using DSC-SEIKO
SII nanotechnology.
2.3.5. Estimation of total sugar content
Total polysaccharide content was determined using phenol
sulphuric acid method [22–24].
In hot acidic medium polysaccharide is dehydrated to hydroxyl fur-
fural. This forms a coloured product with phenol and has absorption
maximum at 488 nm.
A) Preparation of blank solution
To 1 mL of distilled water, 1 mL 5% phenol was added followed by
5 mL of concentrated H2SO4.
B) Preparation of standard solution
A stock solution 100 μg/mL of glucose was prepared in distilled
water. Aliquots were taken from this solution to obtained sugar concen-
tration 60–90 μg/mL. 1 mL of 5% phenol solution was added to 1 mL of
Table 3
Total aerobic count of Cordia gum powder.
Sr. no. Total aerobic count
1 For bacteria
2 For fungi
sugar solution followed by 5 mL of concentrated H2SO4. The absorbance
was measured after standing for 10 min at 488 nm against blank.
C) Estimation of polysaccharide in Cordia dichotoma Gum (test
preparation)
100 mg gum powder was treated with 5 mL 2.5 N HCl and boiled on
a water bath for 3 h. Solid sodium carbonate was added to the resultant
cooled solution after 3 h till the effervescence ceases. Volume was made
up to 100 mL with distilled water. The hydrolysed gum solution was
then centrifuged and 1 mL solution was diluted to 10 mL to get 100
μg/mL solution. 1 mL of this diluted solution was added to 1 mL of 5%
phenol solution followed by 5 mL of concentrated H2SO4. The absor-
bance was measured after 10 min at 488 nm against blank using UV–
visible spectrophotometer. The experiment was carried out in triplicate.
2.3.6. Investigation of structure of the polysaccharide by FTIR
IR spectrum of the polysaccharide was taken using a Fourier trans-
form infrared spectrometer (FTIR, SHIMADZU). C. esculenta gum was
ground with potassium bromide (KBr) powder and then pressed into
1 mm pellets for FTIR measurement.
2.3.7. Rheological study of Cordia dichotoma gum
Rheological studies were performed using Brookfield viscometer. 1%
w/v solution of the gum was prepared and after 48 h of soaking dial
readings were taken using Brookfield viscometer at different rpm for
5 min using spindle no. 2. Graphs were plotted of shear stress and vis-
cosity against shear rate.
2.3.8. Microbial limit test
The total microbial count of the isolated gum sample was deter-
mined by Plate count method as per Indian Pharmacopoeia [25].
A) Preparation of Inoculums
1 g powder of mucilage or polymer was suspended in 10 mL of ster-
ile water (inoculum). 1 mL of inoculum was transferred to 99 mL dilu-
tion blank (sterile water) which was diluted inoculum.
B) Plate Count Technique
i) For Bacteria
1 mL pre-treated sample preparation was added to 15 mL of lique-
fied Casein Soybean digest agar at 45 °C. This mixture was then trans-
ferred to petri dish and allowed it to solidify. Two such Petri dishes
were prepared using same dilution and incubated at 30 °C to 35 °C for
5 days. These were then observed for growth of bacteria and the num-
ber of CFU/g was counted.
ii) For Fungi
Proceeded as described in the test for bacteria but used Sabouraud's
dextrose agar with Chloramphenicol in place of Casein Soyabean Digest
agar and plates were incubated at 20–25 °C for 5 days and then ob-
served for growth of fungi and the number of CFU/g was counted.
3. Results and discussion
3.1. Isolation and characterization of Cordia dichotoma polysaccharide
No. of CFU/g
140 The polysaccharide obtained was dark brown coloured, odourless,
80
tasteless and smooth in texture (Yield = 25% w/w). The result of
 H.A. Pawar et al. / International Journal of Biological Macromolecules 115 (2018) 871–875
physicochemical evaluation of Polysaccharide are summarised in
Table 1 and preliminary phytochemical test are in Table 2.
The angle of repose, tapped density, bulk density and Carr's capacity
data reveals that Cordia dichotoma polysaccharide possesses good flow
properties. Therefore, it can be used in the tablet formulation prepared
by direct compression technique. The pH of Cordia gum in solution
form is close to neutral. This indicates Cordia gum may not cause any ir-
ritation to the epithelium and mucous membrane in the GI tract. The
swelling capacity of Cordia gum was found to be high. Based on the
physicochemical studies, Cordia dichotoma gum could be used as a phar-
maceutical excipient. The substantial swelling shown by Cordia
dichotoma polysaccharides may be useful for achieving controlled drug
release behavior from polysaccharides matrix.
DSC thermogram of Cordia dichotoma polysaccharide exhibited
broad endothermic peak at about 119.5 °C (Fig. 1) due to loss of free
or bound water present in the polysaccharide. It can be concluded that
glass transition temperature (Tg) of the isolated polysaccharide may
be above 305 °C and hence gum would be stable at normal operational
temperatures.
The standard curve of glucose for phenol sulphuric acid method for
determination of total polysaccharide content of Cordia dichotoma
gum is shown in Fig. 2. The calibration curve was found to be linear in
the range 60–100 μg/mL and showed correlation coefficient of 0.9936
indicating good linearity. The regression equation was used for calculat-
ing total polysaccharide content of the polymerisolate by taking average
of three determinations. The total polysaccharide content of Cordia
dichotoma polymer isolate was found to be 86.24% (w/w).
A FTIR spectrum of C. dichotoma is depicted in Fig. 3 the peak at
1170.57 cm−1corresponds to bending vibrational modes of C\\O, pres-
ent in the pyranose ring, while the peaks 1022.28 cm−1 and
862.13 cm−1 is a characteristic contribution of C–OH bending. The
broad bands around 2929.87 cm−1 and 28,525 cm−1 are attributed to
C\\H stretching and to O\\H stretching vibration, respectively. The
broad peak at 1728 cm−1 is a characteristic of C_O stretch of carboxylic
acid which revealed that some amount of uronic acid may be present in
Cordia dichotoma gum. It has been reported in literature that Cordia
dichotoma gum is a polyuronide consisting of arabinose, galactose, and
glucoronic acid in the proportion of 10:7:2 mol; rhamnose is present
in traces [26]. The FTIR data obtained supports the literature results.
The rheological properties of Cordia dichotoma polymer were studies
by plotting rheograms. Fig. 4 shows rheogram of shear stress versus
shear rate. It was found that the curve obtained from measurement at
decreasing shear rate super imposed on that obtained from measure-
ments taken at increasing shear rate.
Fig. 5 shows rheogram of viscosity versus shear rate. It was found
that viscosity decreases with increasing shear rate. The polymer solu-
tion showed shear thinning flow but exhibited Newtonian behavior at
extreme shear rates, both at low as well as high shear rates. The regions
where the viscosity seems to be constant are known as Newtonian re-
gions and the region between these extremes is known as Power-Law
region [27].
The total aerobic count for bacteria and fungi by plate count method
is shown in Table 3.
4. Conclusion
It was concluded that the polysaccharide isolated from Cordia
dichotoma fruits showed the presence of carbohydrates and was found
acceptable for all the tested organoleptic properties. Based on the phys-
icochemical, microbiological limit test, phytochemical studies, it may be
pointed out that the polysaccharide isolated from Cordia dichotoma
875
fruits has the required properties and could be used as an excipient
for pharmaceutical dosage forms.
Acknowledgement
Authors are very much thankful to Dr. P. S. Gide, Principal of Hyder-
abad Sindh National Collegiate Boards (HSNCB's) Dr. L. H. Hiranandani
College of Pharmacy, Ulhasnagar for his continuous support, guidance
and encouragement.
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