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101
Current Drug Therapy, 2016, 11, 101-114
RESEARCH ARTICLE
ISSN: 1574-8855
eISSN: 2212-3903

Glycyrrhetinic Acid Ammonium Loaded Microspheres Using

Colocasia esculenta and Bombax ceiba mucilages: In Vitro and
In Vivo Characterization

Sharad Vishta,b,* and Giriraj T. Kulkarnic

a
Department of Pharmaceutical Science, Jawaharlal Nehru Technological University, Kukatpally
500085, Hyderabad, India; bDepartment of Pharmaceutical Technology, Meerut Institute of Engineering
and Technology, NH-58, Bypass Road-Baghpat Crossing, Meerut-250005, Uttar Pradesh, India;
c
Amity Institute of Pharmacy, Amity University, Sector 125, Noida 201303,Uttar Pradesh, India

Abstract: Background: Glycyrrhetinic acid ammonium (GAA) inhibits the

ARTICLE HISTORY
Received: October 19, 2015
Revised: March 28, 2016
Accepted: April 12, 2016
DOI:
10.2174/15748855116661609070944
25
Current Drug Therapy
biosynthesis of prostaglandins PGE-2 and PGF-2α to their inactive
metabolites. This causes an increased level of prostaglandins in the digestive
system which inhibit gastric secretion.
Objective: The aim of the present investigation was to develop mucoadhesive
microspheres of GAA using Colocasia esculenta (CE) and Bombax ceiba (BC)
mucilages, alone and in combination for the efficient treatment of gastric ulcer.
Methods: The glycyrrhetinic acid (GA) was extracted as ammonium salt
from dried stolons of Glycyrrhiza glabra using hot maceration technique.
Extraction of mucilage was carried out using herbs and rhizomes of CE and flowers of BC by
cold maceration method. The hydrophilic molecule of GAA was encapsulated using single phase
emulsification technique. Glutaraldehyde was used as cross-linking agent. The developed micro-
spheres were evaluated for morphology, particle size, entrapment efficiency, drug loading, in
vitro mucoadhesion, swelling behavior, in vitro drug release and in vivo ulcer healing activity.
Results: The results showed that the drug-polymer in 1: 2 ratio in CE- mucilage containing
microspheres exhibited prolonged drug release as compared to BC-mucialge or their
combination. The stability study results showed stable character of GAA in the developed

microspheres. The developed microspheres show effective in vivo ulcer healing activity in
Wistar male albino rats.
Conclusion: The preliminary results of this study suggest that the developed mucoadhesive
microspheres containing GAA could be a promising tool in the treatment of gastric ulcer and
can be advantageous over the conventional anti ulcer therapy. The study also suggested that the
extracted mucilages can be used in the preparation of the mucoadhesive microspheres.
Keywords: Bombax ceiba mucilage, Colocasia esculenta mucilage, glycyrrhetinic acid, microspheres, mucoadhesive.

INTRODUCTION important part of such novel carrier based drug

Multiparticulate carrier systems offer an
intelligent approach for drug delivery by coupling
the drug to a carrier particle which modulates the
drug release profiles. Microspheres form an
*Address correspondence to this author at the Department of
Pharmaceutical Technology, Meerut Institute of Engineering and
Technology, NH-58, Bypass Road-Baghpat Crossing, Meerut-
250005, U.P., India; Tel: +91-9719302784;
E-mail: sharadvishtpharma@gmail.com
2212-3903/16 $58.00+.00
delivery systems due to their small size [1, 2].
Microspheres are ideal vehicles for many
controlled drug delivery applications due to their
ability to encapsulate a variety of drugs,
biocompatibility, high bioavailability and
sustained drug release characteristics [3].
However, the success of such carrier systems is
limited for the delivery of drugs having narrow
absorption window due to their short residence
time at the site of absorption. This can be
©2016 Bentham Science Publishers
102 Current Drug Therapy, 2016, Vol. 11, No. 2
overcome by developing mucoadhesive micro-
spheres. Conferring mucoadhesive properties to
microspheres will increase intimate contact with
the mucosal membrane, which leads to the
prolonged retention of dosage form in the
gastrointestinal tract, which is the foremost
requirement in the treatment of gastric ulcer [4-9].
Gastric ulcer is a small erosion of gastric
mucosa due to the imbalance between aggressive
and defensive factors. Stomach defends itself from
pepsin and hydrochloric acid either by the
production of a mucus coating on stomach tissue
or by producing bicarbonate. According to Lucas,
the earliest evidence of the application of licorice
in medicine comes from the stores of licorice
found in the ancient tombs of Egyptian Pharaohs
[10]. It is obtained from dried, peeled or unpeeled
roots and stolen of Glycyrrhiza glabra Linn,
(Leguminosae) [11, 12]. Its chief constituent is
triterpenoid saponin, glycyrrhizin, which hydrolyzes
in acidic media and yields one molecule of
glycyrrhetinic acid (aglycon) and two molecules of
glucuronic acid (glycon). Glycyrrhetinic acid (GA)
protects the stomach by stimulation of gastric
mucus production. It enhances the rate of
incorporation of various sugars into gastric
mucosal glycoproteins, promotes mucosal cell
proliferation, inhibits mucosal cell exfoliation,
inhibits prostaglandin degradation, increases the
release of PGEs, reduces the formation of
thromboxane B2 and regulates DNA and protein
synthesis in gastric mucosa. It does not inhibit acid
secretion. GA increases mucus production, which
remain adhered to the gastric mucosa [13, 14].
Other action includes prolongation of life span of
gastric epithelial cell, prevention of bile reflux and
slowing of prostaglandin degradation in gastric
mucosa.
Colocasia esculenta (CE) is a perennial herb
that belongs to the Araceae family. Colocasia
esculenta originates from the tropical region
between India and Indonesia and has been grown
in the South Pacific for hundreds of years. Bombax
ceiba (BC) is a tree of the genus Bombax and it is
commonly known as cotton tree or Red Silk-
Cotton or Red Cotton Tree. The dry cores of the
Bombax ceiba flower contain mucilage. The
present work was aimed to formulate and
characterize mucoadhesive microspheres of
glycyrrhetinic acid ammonium (GAA) using
mucilages obtained from natural sources in order
Visht and Kulkarni
to improve its oral absorption and to decrease the
drug administration frequency.
MATERIALS AND METHODS
Materials
The dried stolons of Liquorice (Glycyrrhiza
glabra) were purchased from the local market of
Meerut, India. The flowers of Bombax ceiba and
rhizome of Colocasia esculenta were procured,
respectively, from Meerut and Rudraprayag, India.
The herbs were authenticated at the National
Bureau of Plant Genetic Resources (NBPGR), New
Delhi. Pure Glycyrrhetinic acid (Sigma Aldrich)
was purchased from RK Enterprises, Meerut,
India. Carbenoxolone sodium was purchased from
Aar Vee and Company, Meerut, India. All the
chemicals used were of analytical grade and used
as such without further purification.
Methods
Extraction of Glycyrrhetinic Acid as its
Ammonium Salt
Glycyrrhetinic acid (GA) was extracted from
dried stolons of Glycyrrhiza glabra. Briefly,
accurately weighed 100 g liquorice powder
(Glycyrrhiza glabra) was soaked in 500 ml pre-
acidified distilled water. This leads to the
hydrolysis of ether bond of glycyrrhizin resulting
in one molecule of aglycon i.e., glycyrrhetinic acid
and two molecules of glycon, i.e., glucuronic acid.
The strong ammonia solution was added to the
mixture and filtered. Activated charcoal was used
for de-pigmentation. The product was crystallized
and purified by column chromatography using
chloroform: methanol (1:1) as mobile phase [15].
Identification of GAA
Isolated and purified GAA was characterized
for physical characteristics, organoleptic properties,
melting point, loss on drying and pH. Elemental
analysis was performed to ensure the counter ions.
The calibration curve of GAA was prepared in 0.1
N HCl using a UV spectrophotometer (1700S,
Shimadzu, Japan). The Fourier Transform Infrared
Spectroscopy (FTIR) (8400S, Shimadzu, Japan)
was performed in order to confirm the molecular
entity using KBr disk method. The sample was
gently triturated with KBr powder at a weight ratio
of 1:100 and compacted into a disc using a KBr
Glycyrrhetinic Acid Ammonium Loaded Microspheres Current Drug Therapy, 2016, Vol. 11, No. 2 103

press at 10 tons for 2 min. The sample disc was
scanned from 4000 to 400 cm-1 at a resolution of 4
cm-1. Reverse Phase High Performance Liquid
Chromatography (HPLC) (8400S, Shimadzu,
Japan) was carried out using C-18 column.
Acetonitrile: phosphoric acid (3:1), pH 2.5, was
used as mobile phase at a flow rate of 0.5 ml/min
using a UV detector at 252 nm. Dilutions of the
sample were prepared in chloroform. The injection
volume was kept at 20 μl.
Extraction of Mucilages
The mucilages were extracted from the herbs of
Colocasia esculenta (rhizomes) and Bombex Ceiba
(red flowers) using cold maceration method.
Acetone was used as precipitation solvent [16-19].
The extracted mucilages were washed three times
with ethanol to decrease microbial load.
Identification of Mucilages
Isolated mucilages were characterized for
physical characteristics, organoleptic properties
and loss on drying. The chemical tests were
performed to find out the presence of unwanted
chemicals, if any [20]. The Fourier Transform
Infrared (FTIR) spectral data of mucilage were
taken for the identification of mucilage by the KBr
disc method using FTIR spectrophotometer
(8400S, Shimadzu, Japan).
Formulation of Mucoadhesive Microspheres
Mucoadhesive microspheres containing GAA
were prepared using CE and BC mucilages alone
and in combination using the single phase
emulsification technique (Table 1). An accurately
weighed amount of GAA was dissolved in 25 ml
of freshly prepared and cooled distilled water. The
mucilage was added with constant stirring for 12 h,
at 1500 rpm using a mechanical stirrer (RQT-124A,
Remi, Mumbai, India), to swell the mucilage. The
mixture was dispersed slowly in 250 ml of
preheated (40°C) light paraffin at 1500 rpm using
a mechanical stirrer for 12 h. Glutaraldehyde
solution (2%) in methanol (cross-linking agent)
was added drop wise to the resultant mixture. The

Table 1. Composition of microspheres.

Amount (mg)
Formulation Code D:P P1:P2 Mucilage RPM
Drug (mg)
P1 P2

A 1:1 - 1500 1500 0 1500

B 1:1 - 1500 0 1500 1500

C 1:2 - 1000 2000 0 1500

D 1:2 - 1000 0 2000 1500

E 2:1 - 2000 1000 0 1500

F 2:1 - 2000 0 1000 1500

G 1:1 1:1 1500 750 750 1500

H 1:1 1:2 1500 500 1000 1500

I 1:1 2:1 1500 1000 500 1500

J 1:2 1:1 1000 1000 1000 1500

K 1:2 1:2 1000 666.66 1333.33 1500

L 1:2 2:1 1000 1333.33 666.66 1500

M 2:1 1:1 2000 500 500 1500

N 2:1 1:2 2000 333.33 666.66 1500

O 2:1 2:1 2000 666.66 333.33 1500

*P1 and P2 indicates mucilage of C. esculenta and B. ceiba, respectively.
104 Current Drug Therapy, 2016, Vol. 11, No. 2
system temperature was maintained at 40°C
throughout the process. Microspheres were
collected and washed 5 times with n-hexane and
petroleum ether. Finally, the mixture was
centrifuged at 2000 rpm and the microspheres
were spread on petri dish to dry in hot air oven for
2 h at 50°C [21].
CHARACTERIZATION OF MICROSPHERES
Determination of Size and Morphology
The particle size of prepared microspheres was
determined by an optical microscopic method
using Quasmo electronic microscope, equipped
with an ocular micrometer and a light camera
(Seagull DF-1, China). The dried microspheres
were placed on a glass slide and two hundred
particles were randomly observed for the
determination of mean diameter and size
distribution. Samplings were done in triplicate for
each formulation [22]. The surface morphology of
developed microspheres was analyzed using
Scanning Electron Microscopy (SEM) (CARL
ZEISS AG-EVO®40 Series) using Thermo Ultra
Dry SDD EDS detector. The samples for SEM
analysis were mounted on brass stub and coated
with gold under an argon atmosphere using a high-
vacuum evaporator (Polaron SEM coating system)
before observation. The photomicrographs were
taken at different magnifications using an
excitation voltage of 20 kV [23].
GAA-Excipients Compatibility Study
The molecular interaction between the GAA
and mucilages was investigated using FTIR
spectroscopy by KBr disc method (8400S,
Shimadzu, Japan). The microspheres were gently
triturated with KBr powder at a weight ratio of
1:100 and then compacted into a disc using a KBr
press at 10 tons for 2 min. The disc was placed in
the sample holder and scanned from 4000 to 400
cm-1 at a resolution of 4 cm-1.
Determination of Entrapment Efficiency and
Drug Loading
Accurately weighed (100 mg) microspheres
were crushed and dispersed in 100 ml phosphate
buffer (pH 7.4) for 24 h. After 24 h, the dispersion
was sonicated for 30 min using bath sonicator and
centrifuged at 4200 rpm for 5 min, and analyzed at
252 nm. The encapsulation efficiency and percent
drug loading were calculated as follows [24]:
Visht and Kulkarni
Encapsulation efficiency (%) = (Calculated
drug concentration)/ (Theoretical drug content) ×
100
Drug loading (%) = (Calculated drug
concentration)/ (Total weight of microspheres) ×
100
Swelling Study
The swelling behaviour of microspheres was
determined by soaking the microspheres in 0.1 N
HCl (pH 1.2) at 37ºC for 24 h. After 5 h, the
microspheres were removed and weighed after
drying the excess water using filter paper. The
percentage swelling index was calculated as
follows [25]:
Swelling index (%) = (Weight of microspheres
after swelling-Initial weight of microspheres)/
(Weight of microspheres after swelling) × 100
Determination of In Vitro Mucoadhesion
Property of Microspheres
The in vitro mucoadhesion property of
microspheres was determined by a falling liquid
film method using freshly excised rat stomach
mucosa (2 x 1 cm). The stomach mucosa was
mounted onto the glass slide using rubber bands. It
was then rinsed with 2 ml of 0.1 N HCl (pH 1.2).
Then weighed amount of microspheres were
hydrated with water and dispersed onto the tissue
specimen. For proper interaction between polymer
and membrane, the glass slide was allowed to
incubate for 15 min in desiccators at 90% relative
humidity. The side was kept at 45º angle relative
to the horizontal plane. The mucosa was
then rinsed with 0.1 N HCl (pH 1.2) maintained
at 37ºC for 5 h at a rate of 10±2 ml/ min. The
microspheres remaining on the tissue surface were
collected after 5 h. The residual amount of
medium was separated by centrifugation followed
by drying at 50ºC. The mucoadhesion strength
of the microspheres was calculated as follows
[26]:
Mucoadhesion (%) = (Weight of sample -
Weight of detached particles)/ (Weight of sample)
× 100
In Vitro Drug Release Study
The in vitro release of GAA from the different
formulations was examined using USP type I
apparatus (Electrolab, TDT-08L, Mumbai, India).
Glycyrrhetinic Acid Ammonium Loaded Microspheres
Dissolution medium (0.1 N HCl, pH 1.2, 900 ml)
was filled in the dissolution vessel and stirred at 50
rpm. The temperature was maintained at 37 ±
0.5°C. Microspheres equivalent to 100 mg of
GAA were placed in the dissolution vessel, and the
basket was rotated at 100 rpm. Aliquots were
withdrawn every 15 min in the first hour and then
every hour till the 4th hour, followed by the 6th,
8th, 10th, 12th, 14th and 24th h. Same amount of
medium was transferred to the dissolution vessel
in order to maintain the sink conditions. The
samples were analyzed by a UV spectro-
photometer (UV - 1700, Shimadzu) at 252 nm
after suitable dilution. The study was conducted in
triplicate [23, 27-29]. The drug release data of
formulation C was analyzed for release kinetics
using BIT 1.12 software [30].
In Vivo Ulcer Healing Activity of Microspheres
Animals Used
In vivo antiulcer study to ascertain the efficacy
of formulated microspheres (formulation C) was
carried out in male Wistar albino rats weighing
around 200 g. The animal experimental protocol
was approved by the Institutional Animal Ethical
Committee (No. 711/02/a/CPCSEA). The animals
were handled as per the guidance of the
Committee for the Purpose of Control and
Supervision on Experimental animals (CPCSEA),
New Delhi, India. The animals were housed in
polypropylene cages and maintained at 24°C ±
2°C under a 12 h light/dark cycle and were fed
with ad libitum with standard pellet diet and had
free access to water [31].
Acetic Acid Induced Ulcer Model
The ulcer was induced by injecting 0.05 ml of
30% acetic acid into the gastric wall of the rat. The
animals were divided into four groups (n = 6) as,
Group 1: control (treated with saline solution);
Group 2: standard, treated with cabenoxolone
sodium; Group 3: treated with pure GAA in 1%
CMC; and Group 4: treated with GAA
mucoadhesive microspheres (formulation C). A
small incision was made in the abdomen and
stomach and exposed, followed by injection of
0.05 ml of 30% acetic acid into the gastric
submucosal layer of a glandular portion of the
stomach. The cabenoxolone sodium, pure GAA
and mucoadhesive microspheres were administered
orally at a dose of 100 mg/kg body weight once
Current Drug Therapy, 2016, Vol. 11, No. 2 105
daily for two weeks. After two weeks all the
animals were anaesthetized using anesthetic ether
and sacrificed to measure/score the lesions of
ulceration. The percent of ulcer protection was
determined as follows [32]:
Protection (%) = (Control mean ulcer index -
Test mean ulcer index)/ (Control mean ulcer
index) × 100
Statistical analysis of data obtained from in vivo
ulcer healing activity (area and percentage
inhibition) was carried out by One-way analysis of
variance (ANOVA) followed by Tukey's multiple
comparison test.
Stability Study
In the present work, stability study of the
selected formulation (formulation C) was
performed after storing the microspheres at
25±2°C/60±5% RH, 30±2°C/65±5% RH,
40±2°C/75±5% RH for 6 months in screw capped
amber colored glass bottles. After 1, 3 and 6
months, the microspheres were evaluated for
colour change and percent drug content. The initial
drug content was considered as 100%.
RESULTS AND DISCUSSION
Characterization of GAA
The percentage yield of GAA was found to be
5.23% w/w. Isolated GAA was crystalline, white,
odorless powder, and had characteristic taste. The
melting point, loss on drying, and pH of isolated
GAA were 293±0.5°C, 0.1%, and 4.2±0.01,
respectively. The results of elemental analysis
indicated the presence of ammonium ion as
counter ion. The λmax for GAA was observed at
252 nm in 0.1 N HCl (pH 1.2). The equation of
the line for calibration curve was Y = 0. 0012X,
r² = 0.9987. The Fourier Transform Infra Red
(FTIR) Spectroscopy showed that principle peaks
obtained in the FTIR spectrum of extracted GAA
were in compliance with the standard spectra of GA.
Standard GA, extracted GA and GAA showed
the presence of several bonds like C-C and C-O
between 1300-800 cm-1, C=C and C=O between
1900-1500 cm-1, C-H and O-H between 3800-2700
cm-1. FTIR spectrum of GA (standard), GA
(extracted) and GAA are shown in Fig. 1. The
results of HPLC analysis for the extracted GAA
showed retention time (rt) of 7.3 min (Fig. 2).
106 Current Drug Therapy, 2016, Vol. 11, No. 2 Visht and Kulkarni

80
%T
70
%T 70
60
60
50
50
40
40
30
30
20
GA Std 20 GA Extracted

4000 3500 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 400 4000 3750 3500 3250 3000 2750 2500 2250 2000 17501500 1250 1000 750 500
1/cm 1/cm
80
%T
40
70 %T
35
60
30
50 25
20
40
15
30
10
20 GA A 5 B. ceiba mucilage

4000 3500 3000 2500 2000 1500 1000 500

1/cm 4500 4000 3500 3000 2500 2000 1750 1500 1250 1000 750 500
1/cm

67.5
120 %T
%T 60
105
52.5
90
45
75
37.5

60 30

45 C. esculenta mucilage 22.5 GAA+ B. ceiba mucilage microspheres

4500 4000 3500 3000 2500 2000 1750 1500 1250 1000 750 500 1/cm 4500 4000 3500 3000 2500 2000 1750 1500 1250 1000 750 500
1/cm
90
%T 67.5
80 %T
60
70
52.5
60
45
50
37.5
40
30
30 22.5
GAA+ C. esculenta mucilage microspheres GAA+ C. esculenta + B.ceiba mucilage microspheres

4500 4000 3500 3000 2500 2000 1750 1500 1250 1000 750 500 1/cm 4500 4000 3500 3000 2500 2000 1750 1500 1200 1000 750 500 1/cm

Fig. (1). FTIR of GA (standard), GA (extracted), GAA, BC mucilage, CE mucilage, GAA + BC mucilage microspheres, GAA
+ CE mucilage microspheres, GAA + CE mucilage + BC mucilage microspheres.

Characterization of Mucilages Pink color was obtained when an aqueous solution

The percentage yield was found to be 9.23%
and 5.36% for CE and BC mucilages, respectively.
The isolated mucilages were brown in color,
odorless, tasteless and had a 0.1% loss on drying.
of each mucilage was treated with ruthenium red
solution, indicates the presence of mucilage. This
was also supported by the formation of violet red
color when an aqueous solution of mucilage was
Glycyrrhetinic Acid Ammonium Loaded Microspheres Current Drug Therapy, 2016, Vol. 11, No. 2 107

203.7 43

Signal [mV]
152.8 20
Area [mAS]

101.8 -3

50.9 -26

-49
0
0 2.0000 4.0000 6.0000 8.0000 10.0000 00:00 02:00 04:00 06:00 08:00 10:00
Quality Time [mm:ss]

Fig. (2). HPLC analysis of GAA.

treated with thionine solution followed by washing
with alcohol, indicating presence of mucilage. The
results of chemical tests performed on both the
mucilages confirmed absence of starch, alkaloids,
saponins and tannins (Table 2). CE mucilage was
more viscous than BC mucilage. FTIR spectrum of
BC mucilage and CE mucilage are presented in
Fig. (1). The mucilages showed the presence of
lactone at 1734 cm-1, -OH between 3400-3200 cm-
1
, -COOH between 1574-1557 cm-1, -CH3 at 2923
cm-1, -OH between 3609-3288 cm-1, ether linkage
between 1455-1400 cm-1, -CH stretching between
2923-2854 cm-1, -C-O stretching at 1018 cm-1, and
-CH3 at 2923 cm-1.
Table 2. Results of chemical tests of extracted CE and
BC mucilage.
Test
Alkaloids
Carbohydrates
Saponins
increase in the particle size was observed with an
increase in polymer concentrations, which might
be due to the more viscous nature of polymer
solutions at higher concentrations (Table 3). The
developed microspheres had a spherical shape
with a smooth surface. The SEM micrographs
presenting morphological characteristics are
shown in Fig. (3).
Drug-Excipients Compatibility
GAA-BC mucilage microspheres, GAA-CE
mucilage microspheres and GAA microspheres
containing combination of BC and CE mucilages
are presented in Fig. (1). No sign of any loss or
generation of any characteristic peak in the FTIR
spectrum of formulations was observed on a
comparison with the FTIR spectrum of pure GAA
and mucilages. This suggested that all the process
Remark* conditions were relevant to the formation of
- microspheres and which in turns showing the
absence of any kind of interaction in between the
-
GAA and mucilages.
-

Phenolic compounds -
Entrapment Efficiency and Percent Drug
Loading
Glycoside test -
It was observed that the entrapment efficiency
Proteins and amino acids -
was increased with an increase in polymer
Phytosterols - concentration. This might be due to the increased
Fixed oils and fats -
viscosity of polymer solutions, which brought
about the increase in the emulsification leading to
Mucilage + the increase drug entrapment (Table 3). The drug
*- Indicates absence, + indicates presence. entrapment was more in microspheres containing
CE mucilage, which might be due to the increased
Characterization of Microspheres viscosity of the dispersion, which limits stripping
of the drug from microspheres. Drug loss in
Particle Size and Morphology continuous phase occurs while the dispersed phase
The mean size range of microspheres was in the stayed in a transitional, semisolid state i.e. mostly
range of 4-12 μm for all the formulations. An during the first 10 min of particle formation. If the
108 Current Drug Therapy, 2016, Vol. 11, No. 2 Visht and Kulkarni

Table 3. Evaluation parameters of different formulations of microspheres (mean ± SD, n = 3).

Formulation Practical Yield Entrapment Loading Swelling Muaoadhesion Mean Particle

Code (mg) (%) (%) (%) (%) Size (µm)

A 2995.74±0.32 99.75±0.54 49.88±0.12 78.06±0.42 71.46±0.15 9

B 2996.39±0.21 99.81±0.63 49.90±0.32 76.81±0.15 70.34±0.74 8

C 2994.73±0.12 99.73±0.25 33.24±0.65 82.34±0.49 73.43±034 10

D 2995.78±0.43 99.70±0.69 33.23±0.45 79.36±0.23 72.65±0.95 12

E 2996.97±0.26 99.85±0.58 66.57±0.25 71.29±0.24 63.23±0.45 6

F 2997.14±0.31 99.89±0.45 66.60±0.36 69.74±0.31 62.45±0.21 7

G 2996.34±0.17 99.85±0.15 49.93±0.14 74.12±0.32 65.34±0.61 4

H 2995.32±0.35 99.81±0.34 49.90±0.42 73.25±0.33 64.12±0.21 12

I 2996.83±0.53 99.84±0.29 49.92±0.43 74.96±0.21 66.32±0.14 7

J 2995.31±0.43 99.74±0.13 33.25±0.16 75.26±0.25 68.31±0.17 6

K 2995.42±0.54 99.80±0.45 33.27±0.74 75.12±0.61 67.23±0.16 8

L 2994.83±0.19 99.72±0.33 33.24±0.17 76.47±0.18 69.32±0.11 8

M 2995.72±0.35 99.84±0..35 66.56±0.16 66.43±0.11 60.43±0.45 4

N 2996.14±0.87 99.85±0.25 66.57±0.15 63.57±0.42 59.34±0.24 5

O 2995.26±0.45 99.75±0.54 66.50±0.22 68.25±0.62 61.42±0.21 6

solubility of the drug in the continuous phase is In Vitro Mucoadhesion

higher than in the dispersed phase, the drug will
easily diffuse into the continuous phase during this
stage [24]. But, in the present study, the drug is
insoluble in the continuous phase (light paraffin)
and the solidification of polymers occurs
immediately when it comes in contact with cross-
linking agent. Hence, the drug will not get diffuse
into the surrounding medium. Both these factors
might be responsible for the increased drug
entrapment.
Swelling Index
Swelling is the major factor which influences
mucoadhesion and the drug release profiles.
Therefore, it is necessary to evaluate the swelling
ability of the developed system. Formulation-C
showed good swelling index (82.34±0.49%) when
compared to the other formulations after 5 h.
Formulation-N exhibited least swelling index
(63.57±0.42%) where the polymer was at its lower
level (Table 3). Formulation C contained only EC
mucilage (more viscous) at higher concentration.
The difference in swelling might be due to
viscosity differences of the mucilages.
The smooth surface allowed higher solvent
penetration into the polymeric matrix, which
produced a viscous gel layer and increased the
mucoadhesion. Higher swelling is generally
associated with higher mucoadhesion, which
might be due to higher interpolation of polymer
chains between microspheres and the mucous
membrane. Formulation-C showed good muco-
adhesive property (73.43±034%) when compared
to the other formulations after 5 h. Formulation-N
exhibited least mucoadhesion (59.34±0.24%). The
results of mucoadhesion study are presented in
Table 3.
In Vitro Drug Release
From the release pattern of all formulations, it
was observed that formulation C showed sustained
drug release over a period of 24 h. Higher
viscosity of CE mucilage might be responsible
for sustained drug release from the microspheres
than BC mucilage containing microspheres. The
mucilage combination does not show any
improved behavior on drug release (Fig. 4). If a
compound is acidic, then it will be more soluble in
Glycyrrhetinic Acid Ammonium Loaded Microspheres Current Drug Therapy, 2016, Vol. 11, No. 2 109

(A) (B) (C) (D)

2 mm EHT = 20.00 kV Data:10 Feb 2011 2 mm EHT = 20.00 kV Data:10 Feb 2011
1 mm EHT = 20.00 kV Data:10 Feb 2011 ZEESS AIRF.JNU ZEESS 2 mm EHT = 20.00 kV Data:10 Feb 2011
AIRF.JNU ZEESS AIRF.JNU AIRF.JNU ZEESS
WD = 11.0 mm Mag = 30.10 kX WD = 10.5 mm Mag = 6.10 kX WD = 10.5 mm Mag = 30.90 kX WD = 10.5 mm Mag = 9.91 kX

(E) (F) (G) (H)

1 mm EHT = 20.00 kV Data:10 Feb 2011 200 mm EHT = 20.00 kV Data:10 Feb 2011 10 mm EHT = 20.00 kV Data:10 Feb 2011 2 mm EHT = 20.00 kV Data:10 Feb 2011
AIRF.JNU ZEESS AIRF.JNU ZEESS AIRF.JNU ZEESS AIRF.JNU ZEESS
WD = 11.0 mm Mag = 17.34 kX WD = 11.0 mm Mag = 55.63 kX WD = 10.5 mm Mag = 4.34 kX WD = 10.5 mm Mag = 4.96 kX

(I) (J) (K) (L)

20 mm EHT = 20.00 kV Data:10 Feb 2011 2 mm EHT = 20.00 kV Data:10 Feb 2011 2 mm EHT = 20.00 kV Data:10 Feb 2011 ZEESS 20 mm EHT = 20.00 kV Data:10 Feb 2011
AIRF.JNU ZEESS AIRF.JNU ZEESS AIRF.JNU AIRF.JNU ZEESS
WD = 10.5 mm Mag = 1.96 kX WD = 10.5 mm Mag = 7.41 kX WD = 10.5 mm Mag = 5.77 kX WD = 10.5 mm Mag = 2.22 kX

(M) (N) (O)

2 mm EHT = 20.00 kV Data:10 Feb 2011 2 mm EHT = 20.00 kV Data:10 Feb 2011 2 mm EHT = 20.00 kV Data:10 Feb 2011
AIRF.JNU ZEESS AIRF.JNU ZEESS AIRF.JNU ZEESS
WD = 10.5 mm Mag = 6.19kX WD = 11.0 mm Mag = 6.63 kX WD = 11.0 mm Mag = 6.54 kX

Fig. (3). Scanning electron micrographs of microspheres showing the general appearance of microspheres (Formulation A-O).
Scales are given on individual micrograph.

basic solutions, and less soluble in acidic dissolution data were plotted according to
solutions. If a compound is neutral (neither acidic Higuchi’s equation, which gives steady state drug
nor basic), then its solubility will be unaffected by release:
pH. The pH of isolated GAA was 4.2±0.01. Q = (D ε/τ) (2Ctot - Cs) Cst1/2
Enhanced solubility of GAA from formulated
microspheres in dissolution medium may be due to where Q is the amount of drug released per unit
the increased surface area of microspheres, area exposed to the solvent, D is the diffusion
swelling and hydration of the polymers. coefficient of the drug in the permeating fluid, ε is
the porosity of the matrix, τ is the tortuosity of the
On the basis of results obtained from the drug matrix, Ctot is the concentration of the solid drug in
entrapment, mucoadhesion, and in vitro drug the dissolution medium, Cs is the saturation drug
release study, formulation C was selected as best and t is the time. Assuming that diffusion
formulation. The drug release data of formulation coefficient and other parameters remain constant
C was analyzed using the zero-order and first- during the release, the above equation reduces to
order equations to determine the drug release [24]:
kinetics from the developed mucoadhesive
microspheres. In the present study, formulation C Q = Kt1/2
was found to exhibit first order release in which Thus, for diffusion controlled release mechanism,
the rate of drug release is dependent of time. For a plot of the cumulative percentage of drug
the further confirmation of the order of release, the released versus square root of time should be
110 Current Drug Therapy, 2016, Vol. 11, No. 2 Visht and Kulkarni

100 100
(A) 90
(B)
Cumulative drug released (%) 90

Cumulative drug released (%)

80 80
A 70
70
60 B 60 F
50 C 50 G
40 D 40 H
30 E 30 I
20 20 J
10 10
0 0
0 100 200 300 400 500 600 700 800 0 100 200 300 400 500 600 700 800
Time (min) Time (min)
100
Cumulative drug released (%)
90 (C)
80
70
60 K
50 L
40 M
30 N
20 O
10
0
0 100 200 300 400 500 600 700 800
Time (min)

Fig. (4). In vitro drug release profile (A, B and C) from various formulations (formulation A-O) in 0.1 N HCl (pH 1.2) at 37°C
(mean ± SD, n=3).

linear. The linearity of the plots was confirmed by value of n is less than or equal to 0.5, the
the calculation of correlation coefficient. mechanism of drug release is diffusion without
swelling. If the value is greater than 0.5 and less
To find out the mechanism of drug release, and
than 1, the release is through diffusion with
also to verify the fact that whether diffusion is
swelling and if it is above 1, the release
Fickian or non-Fickian, the in vitro dissolution
mechanism is anomalous diffusion, not confirming
data of formulation C was plotted according to
to Fick’s laws (non-Fickian).
Peppas’ equation, in which log cumulative
percentage of drug release was plotted against log The n and k values for the Korsemeyer Peppas
time. According to the logarithmic form of equation were 0.95 and 0.63, respectively. From
Peppas’ equation, the rate of drug release can be the kinetic data (Table 4), it is evident that the
expressed as [33, 34]: drug release kinetics was found to follow
Higuchi’s as well as Peppas’ kinetics for all the
log Q = log K + n log t
formulations and the drug release was Anomalous
where Q is the amount of drug released, t is the Transport. The calculated slope values of
time and n is the slope of the linear plot. If the Higuchi’s and Peppas’ equations gave a value

Table 4. Release kinetics parameters of the best selected formulation C.

Hixson Crowell Peppas’ Korsemeyer Peppas’

Zero Order First Order Higuchi’s Matrix
Model Model Model

r2 k r2 k r2 k r2 k r2 k n k

0.7779 0.1336 0.9766 -0.0041 0.9398 10.2534 0.8161 0.6301 0.9247 0.0009 0.9525 0.6301
Glycyrrhetinic Acid Ammonium Loaded Microspheres Current Drug Therapy, 2016, Vol. 11, No. 2 111

Table 5. Comparison of in vivo ulcer healing activity of formulation C. The data represent mean ± SD (n=6).

Treatments Dose (mg/kg) Area (mm2) Inhibition (%)

Control Normal saline solution 341.50±0.63 -

Carbenoxolone sodium 100 mg 80.17±0.23 76.52±0.68

GAA 100 mg 116.98±0.38 65.74±0.94

Formulation C Equivalent to 100 mg of GAA 93.94±0.35 72.49±0.75

Table 6. Results of One Way ANOVA on the in vivo ulcer healing activity of formulation C.

Source of Variation Sum of Square Degree of Freedom Mean Square F Ratio

One Way ANOVA on area

Between 136500 3 45520 254000

Within 1.433 8 0.1792

Total 136500 11

Tukey's multiple comparison test Mean Diff. q Level of significant difference

Control vs Carbenoxolone sodium 261.3 1069 P < 0.001

Control vs GAA 224.5 918.7 P < 0.001

Control vs Formulation C 247.6 1013 P < 0.001

Carbenoxolone sodium vs GAA -36.81 150.6 P < 0.001

Carbenoxolone sodium vs Formulation C -13.77 56.35 P < 0.001

GAA vs Formulation C 23.04 94.28 P < 0.001

One Way ANOVA on percentage inhibition

Between 178.0 2 89.01 61.67

Within 8.660 6 1.443

Total 186.7 8

Tukey's multiple comparison test Mean Diff. q Level of significant difference

Carbenoxolone sodium vs GAA 10.78 15.54 P < 0.001

Carbenoxolone sodium vs Formulation C 4.030 5.810 P < 0.05

GAA vs Formulation C -6.750 9.732 P < 0.01

close to 1 but less than 1, which confirmed that the
release mechanism of GAA from the microspheres
was Fickian diffusion with erosion (anomalous
transport). The results confirmed that the best fit
model was first order, and the drug release
mechanism was anomalous transport.
In Vivo Ulcer Healing Study
In vivo ulcer healing activity was investigated
using Wistar male albino rats. The order of in vivo
ulcer healing activity was as carbenoxolone
sodium > GAA microspheres > GAA. In in vivo
ulcer activity study, the animals treated with GAA
and microspheres (formulation C) showed a
significant (P<0.001) difference in ulceration area
when compared to the control and standard groups
(Tables 5 and 6). The results of in vivo ulcer
healing study are shown in Fig. (5). Compare to
GAA, 1.10 fold (p<0.01) higher inhibition of ulcer
was observed when animals were treated with
formulation C.
112 Current Drug Therapy, 2016, Vol. 11, No. 2 Visht and Kulkarni

Fig. (5). Results of in vivo ulcer healing activity demonstrating effectiveness of Carbenoxolone sodium (A), GAA (B) and
formulation C (C).

Table 7. Results of stability study of microspheres at different storage conditions with variable effect on drug content
(%) and effect on formulation colour (mean ± SD, n=3).

Storage Conditions Time (Months) Colour Change Drug Content (%)

1 No change 99.93±0.13
25±2°C/60±5%RH 3 No change 99.16±0.32

6 No change 98.84±0.11

1 No change 99.87±0.22

37±2°C/65±5%RH 3 No change 99.13±0.21

6 No change 97.58±0.31

1 No change 99.97±0.26

45±2°C/75±5%RH 3 No change 97.54±0.17

6 No change 96.25±0.31

Stability Study stomach for longer duration owing to smaller

particle size of microspheres. Further dose
Effect of storage conditions on color change
administration frequency can be minimized;
and percentage drug content of microspheres were absorption rate will be faster with improved
observed. The initial drug content was taken as solubility as a salt form of glycyrrhetinic acid is
100% and compared with the drug content at 1, 3 used.
and 6 months interval. The results showed that
there was no change in color of microspheres ABBREVIATIONS
after 6 months and the drug content was in the
acceptable range (Table 7). BC = Bombax ceiba
CE = Colocasia esculenta
CONCLUSION
CMC = Carboxy methyl cellulose
From the present study, it can be concluded that FTIR = Fourier Transform Infrared spectro-
GAA can be effectively loaded into mucoadhesive photometer
microspheres using natural polymers. This can be
advantageous over conventional ulcer treatment GA = Glycyrrhetinic acid
approaches, as the dosage form can reside in the GAA = Glycyrrhetinic acid ammonium
Glycyrrhetinic Acid Ammonium Loaded Microspheres Current Drug Therapy, 2016, Vol. 11, No. 2 113

HCl = Hydrochloric acid [11] Dehpour AR, Zolfaghari ME, Samadian T, Kobarfard F,
Faizi M, Assari M. Antiulcer activities of liquorice and its
HPLC = High Performance Liquid Chromato- derivatives in experimental gastric lesion induced by
ibuprofen in rats. Int J Pharm 1995; 119: 133-38.
graphy [12] Isbrucker RA, Burdock GA. Risk and safety assessment on
KBr = Potassium bromide the consumption of Licorice root (Glycyrrhiza sp.), its
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Toxicol Pharm 2006; 46: 167-92.
rpm = Revolution per minute [13] Akiyama Y, Nagahara N, Kashihara T, Hirai S, Toguchi H.
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SEM = Scanning Electron Microscopy prepared for the gastrointestinal tract using polyglycerol
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Pharm Res 1995; 12: 397-405.
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mucoadhesive properties of aminated gelatin microspheres.
The authors confirm that this article content has J Control Release 2001; 73: 223-31.
no conflict of interest. [15] Amani M, Mostofi RSGN, Hosein A, Kasahnai M. Optimal
extraction of glycyrrhetinic acid from Liquorice root. J
Food Technol 2005; 3: 576-80.
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mucilages extracted from Okra fruits (Hibiscus esculentus
The authors are thankful to the Indian institute L.) and Baobab leaves (Adansonia digitata L.). J Sci Food
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