Evaluation of Antacid Activity of Microemulsion Formulation of Blend of Essential Oil

Abstract

Essential oils are having wide range of biological activity is used to achieve therapeutic effects. These are volatile substances sensitive to oxygen, light, moisture and heat. In the present study microemulsion formulation was prepared using a blend of essential oil contains cardamom, coriander, fennel, caraway, ajowan and peppermint oil, water and non ionic surfactant tween 20 and cosurfactant as ethanol. Each essential oil was extracted from dried seed by steam distillation and characterized by Headspace Gas chromatography use of a marker compound which was linalool for coriander oil, cineol for cardamom oil, anethol for fennel oil, carvone for caraway oil, thymol for ajowan oil and menthol for peppermint oil. The marker compound was characterized using mass spectroscopy. Microemulsion of oil showed higher stability with droplet size in the range of 110-410nm. The product then screened for in vitro antacid properties which showed significant positive response.

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163
International Journal of Pharmaceutical Sciences and Drug Research
2015; 7(2): 163-167
Research Article
ISSN: 0975-248X
CODEN (USA): IJPSPP
Evaluation of Antacid Activity of Microemulsion Formulation of Blend of
Essential Oil
Joydeep Mazumder1*, Devender Pathak1, Rachna Kumria2
1Rajiv Academy for Pharmacy, Delhi-Mathura Highway, P.O Chhatikara, Mathura-281 001, Uttar Pradesh, India
2Swift School of Pharmacy, Village- Ghaggar Sarai, Rajpura, Patiala, Punjab, India
ABSTRACT
Essential oils are having wide range of biological activity is used to achieve therapeutic effects. These are volatile
substances sensitive to oxygen, light, moisture and heat. In the present study microemulsion formulation was
prepared using a blend of essential oil contains cardamom, coriander, fennel, caraway, ajowan and peppermint
oil, water and non ionic surfactant tween 20 and cosurfactant as ethanol. Each essential oil was extracted from
dried seed by steam distillation and characterized by Headspace Gas chromatography use of a marker
compound which was linalool for coriander oil, cineol for cardamom oil, anethol for fennel oil, carvone for
caraway oil, thymol for ajowan oil and menthol for peppermint oil. The marker compound was characterized
using mass spectroscopy. Microemulsion of oil showed higher stability with droplet size in the range of 110-
410nm. The product then screened for in vitro antacid properties which showed significant positive response.
Keywords: Essential oil, Microemulsion, Steam distillation, Headspace gas chromatography, Antacid activity.
INTRODUCTION
Essential oils and their main components have many
applications in popular medicine, food, beverages,
preservation, cosmetics as well as in the fragrance and
pharmaceutical industries. [1-2]
Cardamom, Fennel, Coriander, Caraway, ajown and
pippermint is produced from cultivated or wild plants
in the mountainous regions of southern India. These
has been used in the traditional Chinese medicine and
Indian Ayurvedic medicine for thousands of years,
mainly for treating respiratory diseases, fevers and
digestive complaints. These additionally possess
medicinal properties including antibacterial,
*Corresponding author: Mr. Joydeep Mazumder,
Rajiv Academy for Pharmacy, Delhi-Mathura
Highway, P.O Chhatikara, Mathura-281 001, Uttar
Pradesh, India; Tel.: +91-9704577208;
E-mail: joydeeppharma@gmail.com
Received: 12 February, 2015; Accepted: 20 February, 2015
carminative, antioxidant, digestive etc activity. [3-6]
Steam distillation is commonly use technique for
extraction of essential oil from their dried seed or leaf.
The approaches taken for extraction of Cardamom,
Fennel, Coriander, Caraway, ajown and peppermint oil
from their dried seed using Clevenger apparatus steam
distillation apparatus. [7]
The physico-chemical properties of each essential oil
were verified. Each oil was characterized making use of
a marker compound which was linalool for coriander
oil, cineol for cardamom oil, anethol for fennel oil,
carvone for caraway oil, thymol for ajowan oil and
menthol for peppermint oil by a validated Headspace
gas chromatographic method. The marker compound
was confirmed by mass spectroscopy. [8-12]
Essential oil is unstable, volatile and lipophilic in
nature. Microemulsion technology is applied in the
pharmaceutical industry to increase drug penetration
across the diffusion layers, good appearance, drug
solubility and also increase the stability. [13-15]

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Microemulsions are clear, thermodynamically stable,
isotropic liquid mixtures of oil, water and surfactant. In
contrast to ordinary emulsions, microemulsions form
upon simple mixing of the components where two
immiscible phases (water and ‘oil’) are present with a
surfactant, the surfactant molecules form a monolayer
at the interface between the oil and water, with the
hydrophobic tails of the surfactant molecules dissolved
in the oil phase and the hydrophilic head groups in the
aqueous phase.
The aim of the present study is to formulate
microemulsion formulation using a blend of essential
oil, water and non toxic, non ionic surfactant Tween 20
with cosurfactant as ethanol. The formulated product
then screened for the antacid activity. [16-17]
MATERIALS AND METHODS
Material and Reagent
Dried seed of Cardamom, Coriander, Fennel, Caraway,
Ajowan and peppermint oil was procured from trade
market. Markers compounds namely linalool, cineol,
anethol, carvone, thymol and menthol were procured
from Ultra International Limited, Uttar Pradesh, India.
Tween 20 (Polyethylene glycol sorbitan monolaurate)
was procured from Sigma Aldrich, India. Water was
obtained using a Millipore gradient Water System
(Millipore Ltd., Bangalore, India) for all the
experiments. Methanol, Hydrochloric Acid, Sodium
Hydroxide, sodium carbonate and other reagents used
for the analysis were of Analytical grade. The purities
of all the marker standards were not less than 98%.
Extraction of essential oil
Dried seed of cardamom, coriander, fennel, caraway,
ajowan and peppermint oil were collected from the
market. 250 g of each seed boiled with 500 ml of
distilled water in a Clevenger apparatus up to 6 hours.
The volume of essential oils was determined from a
calibrated trap. The essential oils in the distillate were
dried over anhydrous Na2SO4 and kept in the freezer.
The same process has been repeated to get desire
volume.
Physical characterizations, Identification and
estimation of purity of oils
The physico-chemical properties were measured
according to Russian Pharmacopoeia (1990). The color
of the individual oils was checked by visual
observation. Refractive index and optical rotation of the
oils has been checked using Abbe’s Refractometer and
polarimeter. Specific gravity test has been performed
using Borosil Specific Gravity Bottle.
A validated Headspace gas chromatography method
was used to identify & estimate the purity of individual
oil based on marker compound present in each oil
where respective marker compound was used as
standard.
Characterization of marker compounds by Mass
spectroscopy
The individual marker compound was characterized
making use of Atmospheric-Pressure Chemical
Ionization (APCI) mass spectroscopic method to
determine the molecular mass of the marker
compound. This study was carried out at Radiant
Research Center, Bangalore, India.
Preparation of blend of sample:
A blend of oil was prepared by transferred 1g of each
oil in 20 ml volumetric flask. Added approximately
500mg of peppermint oil (used as soothening agent)
into it. Made up the volume with sunflower oil used as
a base. The sample was stored in a close tight amber
color bottle.
Microeuncapslsion Preparation
Microemulsions were formulated using blend of
essential oil, Tween 20, ethanol and water. Tween 20
was selected because it is nontoxic and non ionic in
nature and it has average HLB (Hydrophilic lipophilic
balance) value of 16.7 that is favorable for formulation
oil-in-water emulsion. Ethanol was chosen as
cosurfactant to get better stability and dispersion of the
organic phase into continuous phase.
The surfactant Tween 20 was mixed with cosurfactant
ethanol in 3: 1 ratio (S). Different formulation was
prepared by mixing blend of essential oil (O) with
surfactant-cosurfactant mixer (S) in the ratio of 2:1
(OS1), 2:2 (OS2), 2:3 (OS3) and 2:4 (OS4). The water was
added drop wise externally under continuous stirring
condition using magnetic stir at 300 rpm.
Characterization of Microemulsion
Stability study
Each formulation was centrifuged at 12,000 rpm for 30
min at room temperature to determine their
thermodynamic stability. The physical stability of the
microemulsions like phase separation or creaming was
assessed by visual inspection of the samples stored in
tightly closed tubes at room temperature. The
observation was carried out every day in first week
followed by every week up to 3 months. The test was
performed in triplicate for each sample.
Measurement of pH
Using the Mettler Toledo 320 pH meter, the pH values
of the selected formulation samples were measured at
25±1oC. The measurements were carried out in
triplicate.
Zeta-potential measurement
Zeta potential is a measure of the magnitude of the
electrostatic or charge repulsion/attraction between
particles. The zeta potential is a key indicator of the
stability of colloidal dispersions. A laser doppler
electrophoresis was carried out on the microemulsions
with a Zetasizer Nano Series equipment (Malvern,
Nanoslight NS500), which is capable of measuring sizes
between 10nm to 2000nm. Zeta potential and the
dynamic light scattering (DLS) of the microemulsions
were analysed in duplicate at 25°C.
Assay of microemulsion formulation
In drug product, 1 mL of emulsion contains 2.5 mg of
each oil. A validated Headspace Gas chromatography
assay method was used to as determine content of oil
present in the emulsion against the standard marker

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165
compound. The gas chromatographic system included
a Gas chromatograph with Headspace auto sampler
and a flame ionization detector. A DB-624 Capillary
column with 30 meters length, 0.32 mm ID and 1.8 µm
film thickness was use as a stationary phase. Initial
oven temperature was programmed at 60°C with a
hold for 2 minutes, with a 15°C/ minutes rate. The
temperature was raised to 180°C and hold for 10
minutes. The final temperature was elevated to 240°C
at a rate of 15°C/ minutes. Nitrogen was used as a
carrier gas at flow of 1.2 ml/min. Detector parameter
were programmed as: temperature 270°C, range 1 and
attenuation - 4. Split ratio selected 5:1. The total run
time is 20 minutes. In the head-space the oven
temperature was kept at 85 0C , Needle temperature 95
0C, Transfer line temperature 100°C, GC cycle time 22
minutes, thermostat time 30 minutes, pressurization
time 2 minutes, injection time 0.5 minutes, withdrawal
time 0.5 minutes. Headspace mode was kept constant
and Headspace carrier pressure was fixed to 15 psi.
Both the product and standard (blend of marker
compound) was dissolved in methanol to make the
final concentration 0.5 mg/mL.
Antacid activity studies
Antacid activity evaluation was done using Rossett-
Rice method. The in vitro acid neutralization capacity of
the drug product was evaluate against standard
NaHCO3 and Rossett-Rice time i.e. the time during
which the pH maintained between pH 3.0 and 5.
Three 500 mL glass beaker containing 70 ml 0.1N HCl
and 30ml of distill water was kept on magnetic stirrer.
The electrode of the pH meter was deep into the
solution and temperature of the solution was
maintained 37ºC. 5 mL of drug product containing 12.5
mg of blend of essential oil added into the solution. A
glass burette attached with iron stand filled with 0.1N
HCl and kept on the glass beaker shown Fig. 4. A rate
of 4 ml/min of 0.1N HCl was added into the solution
which is simulates the normal acid secretion rate. The
pH was noted & the Rosette-Rice time was determined.
The test was repeat with 10mL and 20mL of drug
product.
Fig. 1: Typical chromatogram of system suitability solution of standard
Fig. 2: Typical chromatogram of blend sample solution in precision

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Fig. 3: Droplets size distribution of OS4 formulation
Table 1: The table lists the physical properties of the oils
Plant material
Weight (g)
Volume of distilled
water (ml)
Time of heating
(hrs.)
Temperature (ºC)
Volume of oil obtained
(mL)
Cardamom (seeds of the fruit)
250
500
6
80
3.5
Coriander (crushed ripe seeds)
250
500
5
80
2.3
Fennel (crushed seeds)
250
500
5
80
5.0
Caraway (dried ripe seeds)
250
500
5
80
2.0
Ajowan (dried seed)
250
500
5
80
2.5
Table 2: The table lists the physical properties of the oils
Essential oil
Name of Marker
compound
Color
Odor
Relative
density
Refractive
index
Optical
rotation (ºC)
% of oil extracted by
steam distillation (% of
Marker present in oil)
Coriander
Linalool
Colorless to pale
yellow
Characteristic
0.865
1.463
+ 9.003
60%
Caraway
Anethol
Colorless to pale
yellow
Characteristic
0.917
1.485
+ 71.02
61%
Fennel
D-Carvone
Colorless to pale
yellow
Characteristic
0.964
1.545
+ 12.185
65%
Ajowan
Thymol
Colorless to pale
yellow
Characteristic
0.925
1.503
+ 0.446
51%
Cardamom
Cineol
Colorless to pale
yellow
Characteristic
0.922
1.464
+ 26.706
58%
Table 3: Physicochemical characteristics of microemulsions (mean ± SD)
Number of Batch
of formulation OS4
pH
Zeta
Potential (mV)
Range of Droplet
size (nm)
Assay (% of Marker compound present)
Linalool
Anethol
D-Carvone
Thymol
Cineol
OS4-1
6.85 ± 0.02
-39±2
139-395
58%
56%
52%
45%
50%
OS4-2
6.82 ± 0.01
-37±2
125-420
52%
55%
57%
45%
51%
OS4-3
6.80 ± 0.01
-35±4
120-405
56%
58%
54%
47%
55%
OS4-4
6.81 ± 0.03
-36±2
110-370
55%
54%
59%
42%
51%
OS4-5
6.82 ± 0.02
37±3
150-355
54%
60%
51%
45%
48%
RESULTS AND DISCUSSION
The quantity of each essential oil was obtained by
steam distillation was tabulated in Table 1. The
extraction was repeated to obtain required volume of
essential oil. The Physical characterizations of the oils
that odor, color, refractive index and optical rotation
and their purity had done and match with their
specification available in the data bank & literature are
tabulated in Table 2. The purity was established of
blend of essential oil using marker compound of each
oil as standard shown in Fig. 1 and Fig. 2.
Selection and physicochemical characterization of
Microemulsion formulation
The different microemulsion formulations (OS1, OS2,
OS3 and OS4) were prepared were tested for their
phase stability after centrifuge of each formulation.
After centrifuge, immediate phase separation was
observed in OS1 and OS2 formulation but OS3 and OS4
formulation was observed stable and extended their
stability at room temperature. Upon storage for two
week duration, again phase separation was observed in
OS3 but OS4 showed higher stability in entire 3 month

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stability period. Hence OS4 was used for further
characterization and application of studies.
After getting the stable formulation OS4, the process
was optimized by taking 4 different batch formulations
with same formula and evaluate their sameness
characterization properties.
The pH, refractive index, zeta potential, droplets size
and assay for 5 batches of formulation was tested for
evaluation of repeatability of the formulation and
tabulated in Table 3. and Figure 3 shows the droplet
size distribution of OS4 formulation. It is found that all
the 5 batches result are consistence and stable.
Result of Antacid activity
The antacid profile was evaluated by in vitro test
known as Rosette-rise test for 5mL, 10mL and 20mL of
drug product contain 12.5 mg, 25 mg and 50 mg of each
essential oil. It was found that 5mL dose maintained
the pH above 3 for 4.11±0.10 min; 10 mL of dose
maintained the pH 10.10±0.10 min. and 20 mL of dose
maintained the pH 25.10±0.15 min respectively as
compared to standard 2.5 mg NaHCO3 which
maintained the pH for 10.08±.010 min. The drug having
antacid activity should have an adequate duration of
action that can maintain the pH of stomach above 3. 10
mL and 20 mL dose of the product shown in vitro
similar antacid properties and having significant
reactivity towards the acid. Hence the drug product can
be considered as good antacid
From the current study it can reasonable to conclude
that a stable microemulsion of a blend of essential oil
can be prepared by using optimum combination of co-
surfactant ethanol and Tween 20 which has a potential
as a suitable drug delivery system. The results of the
present study recommend that product having
significant acid neutralizing capacity and shown
resistance against change in pH. Hence this product
can be use as antacid to inhibit gastric secretion in the
stomach.
ACKNOWLEDGEMENTS
The authors greatly acknowledge the receipt of pure
marker compound from Ultra International Limited,
Uttar Pradesh, India and Rajiv Academy of Pharmacy,
Mathura, Uttar Pradesh, India for providing research
facilities throughout the project work.
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Source of Support: Nil, Conflict of Interest: None declared.