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Formulation design and evaluation of anti-microbial activity of emulgel

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Int. J. Pharm. Sci. Rev. Res., 40(2), September – October 2016; Article No. 50, Pages: 271-277 ISSN 0976 – 044X

Research Article

Formulation Design and Evaluation of Anti-Microbial Activity of Emulgel Containing Essential Oil of
Myrtus communis L.
a a b c
Hiba Sabah Sabri* , Wedad Kamal Ali , Baydaa Hameed Abdullah , Widad M.K. Al-Ani
a
Department of Pharmaceutics, College of Pharmacy, Al-Mustansiriya University, Baghdad, Iraq.
b
Department of Clinical Laboratory Sciences, College of Pharmacy, Al-Mustansiriya University, Baghdad, Iraq.
c
Department of Pharmacognosy, College of Pharmacy, Al-Mustansiriya University, Baghdad, Iraq.
*Corresponding author’s E-mail: Hiba_sabah86@yahoo.com

Accepted on: 10-09-2016; Finalized on: 30-09-2016.

ABSTRACT
Myrtus communis L (Family Myrtaceae) is an aromatic evergreen perennial shrub or small tree, widespread in the Mediterranean
region, have been used widely in the traditional medicine due to variety of its therapeutic properties. The Extraction of myrtle oil
from the leaves of Myrtus plant by hydrodistillation method using Clevenger type apparatus, produce a light yellow essential oil at
0.55 % yield, the chemical composition of myrtle oil was analyzed by gas chromatography-mass spectrometry (GC-MASS) revealed
that myrtle oil consist of 19 compounds; the major constituents that present in high percentage were: alpha Pinene (32.65.1%),
1,8Cineole (25.1%), Linalool (16.37%), Eugenol (6.56%). The antimicrobial activities of Myrtle oils against Staphylococcus aureus,
Streptococcus pyogen, Pseudomonas aeruginosa and Candida albicans were evaluated by disk diffusion method, Myrtle oil exhibited
varying levels of antimicrobial activity against the investigated pathogens. The objective of this study was to formulate topical
antimicrobial agents containing myrtle oil for the treatment of human skin diseases. The emulgel formulas were prepared by using
different types and concentration of emulsifying agents (Span 80, Tween 80), gelling agents type (methylcellulose, Carbopol940,
Xanthan gum, HPMCK15M) at different ratios and olive oil. The prepared formulas were characterized for the physical appearance,
pH, viscosity and in vitro drug release study. The results revealed that all the prepared emulgel formulas have acceptable physical
attributes. After selection of optimum formula, it was subjected to further tests to confirm its stability, antimicrobial activity, Kinetic
Modeling of drug release and skin irritation study on rat skin.
Keywords: myrtle oil, herbal emulgel, Clevenger type apparatus, emulsifying agent, gelling agent.

INTRODUCTION or stored either in particular region of the plant or in

various organs in the same plants.4 Biological activity of

T opical and transdermal formulations have a long

history of use. Over 2000 years ago, Greek
physicians used formulations containing salt,
vinegar, honey and resins to treat skin lesions and ulcers.
Chinese, Egyptian and Roman medical histories describe
essential oils depends on their chemical composition,
which is determined by the plant genotype and is greatly
influenced by several factors such as geographical origin,
environmental and agronomic conditions.5
numerous remedies applied topically as pastes and External applications of essential oils after mixing with
poultices.1,19 carrier oil involve in massage, lotions, or dressings.
Vaporization and inhalation (taking into the body by
Semisolid preparations represent dosage forms that have
breathing) both are the part of application. Essential oil
properties in between solid and liquid dosage forms and
may be ingredient of gargles and mouthwashes. The
possess characteristic rheological properties such that
internal use of essential oils is very rare. An external
they can be easily applied on biological membranes and
application of the oil is the most common and widely
can be retained on the site of application for a prolonged
used method as compare to the internal application.8
time. Semisolid dosage forms may contain one or more
active ingredients in suspended/dissolved forms, in Myrtus communis L Common myrtle belongs to the
inclusion complexes, or in a solubilized state and are Myrtaceae family, which comprises approximantly 145
applied topically to the skin or on the surface of the eye, genera and over 5500 species. Myrtus communis is an
nasally, vaginally, or rectally for local and/or systemic important medicinal and aromatic plant, because of their
effects.2 Emulgel: Emulsion hydrogels, also known as Essential oils are gaining remarkable interest for their
emulgels, are biphasic hydrogel based formulations. potential multipurpose uses as antioxidant, antibacterial,
Emulgels are usually formed either by dispersing an oil and antiseptic agent.6
phase in a gel phase or by inducing gelation of the
True Myrtle is characterized by its branches, which form a
external phase of an oil-in-water emulsions. The concept
close full head, thickly covered with ovate or lanceolate
of emulgels was conceived to eliminate the limitation of
evergreen leaves. Their leaves are 3–5 cm long and
the hydrogels in delivering hydrophobic drugs.3 Essential
contain tannins, flavonoids and volatile oils.
oils are volatile products of aromatic plants secondary
metabolism, normally formed in special cell or group of This species is a very aromatic plant because of the high
9
cells found in many leaves and stems. It is concentrated essential oil content in its leaf, flower and fruit glands .
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The isolation of essential oils from Myrtus communis L. (Staphylococcus aureus, Streptococcus pyogen,
leaves is usually obtained by hydro distillation method Pseudomonas aeruginosa, and Candida albicans) was
with a Clevenger-type apparatus, according to the Italian confirmed by standard biochemical tests and
Official Pharmacopoeia7. Myrtle oil consists of different morphological studies. Staphylococcus aureus,
types of terpenes, phenols and oxides. The main terpenes Pseudomonas aeruginosa were grown on nutrient agar,
found are monoterpenes, sesquiterpenes, and long chain Streptococcus pyogen was grown on brain heart infusion
terpenes.10 agar, and Candida albicans were cultured on yeast malt
agar plates. Microbial suspensions were then made from
MATERIALS AND METHODS
the agar plates using sterile relevant broths at a
Liquid paraffin and Olive oil were purchased from concentration was adjusted to 0.5McFarland standards,
Solvochem, UK. Carpobol 940, Xanthan gum, Methyl of approximately 106CFU/mL.14,15
cellulose, HPMC K15m, Brij35 and Sabouraud Dextrose
Disc Diffusion Method
Agar Medium were purchased from HIMEDIA, India.
Ethanol was purchased from Sigma Aldrich, USA. Span 80 The fresh oil was tested for its antimicrobial activities.
and Tween80 were purchased from Merck, Germany. The disc diffusion method was used for antimicrobial
Methyl paraben and Propyl paraben were purchased screening as follows:
from Interchimiques SA-France. Triethanolamine was
A Sterile Mueller-Hinton agar medium was used for the
purchased from Alpha Chemika, India. Propylene glycol
antimicrobial assay of Staphylococcus aureus,
was purchased from Avonchem, UK. Sodium sulfate
Pseudomonas aeruginosa. Streptococcus pyogen, and
anhydrous was purchased from BDH-England.
Sabouraud agar for the Candida albicans. The media were
Extraction of Myrtle Oil prepared and allowed to solidify in the plates and then
6
0.1 mL of the microbial suspension (10 CFU/mL) was
until extraction Harvested leaves of Myrtus communis L.
streaked over the surface of the medium using a sterile
were obtained from local Baghdad gardens,, and dried to
glass spreader.31 The well were made by using cork borer
at room temperature then the dried leaves were grinded,
(6mm) under aseptic conditions and then 50 μL from each
and the essential oils of the plant were extracted by
of the oil dilutions were put on each well. The plates were
hydro-distillation method of the plant material, the most
then incubated for 24-48 hours at 37°C in order to get
common method for volatile oil extraction, using a
reliable microbial growth. Microbial inhibition zones were
Clevenger type apparatus.In this process the plant
measured using ruler.18,30
material being boiled in water, using a fire source from
below the vessel. The oils were separated from the Determination of λ max of Myrtle Oil
aqueous layer by using a separatory funnel and
The ultraviolet (UV) absorbance for myrtle oil was
dehydrated with anhydrous sodium sulphate and stored
examined in phosphate citrate buffer solution (pH5.5)
in clean, dark brown bottles at 4°C.This process was
and in absolute ethanol between 200 and 400 nm by UV-
repeated until almost all the essential oil was extracted.11
Visible spectrophotometer and maximum absorption
Identification of Myrtle Oil Components (λmax) values were determined and further used for
plotting calibration curve of the oil in these two media.17
Myrtle oil was analyzed by Gas chromatography/Mass
spectrometry (Shimadzu GCMS-QP2010Ultra), using a Formulation of Myrtle oil Emulgel Formulas
capillary column (HP-5MS, phenyl methyl silicon, 25 m ×
The detailed composition for the emulgel formulas is
0.25 mm, 1 μL injection). Helium was used as the carrier
given in Table 1. The formulations F1 and F2 were
gas (1.53 mL/min).
prepared by dispersing methyl cellulose in heated
The GC oven program was as follows: initial temperature purified water (80°C), and the dispersion was cooled to
70°C for 3 min, programmed rate 12°C/min up to 240°C. room temperature and left overnight to ensure hydration
The injector temperature was 240°C. For MS detection, of the gel While F3 was prepared by dispersing carbomer
an electron ionization system with ionization energy of 70 940 in purified water with continuous stirring, F4, F5, F7
eV was used. Injector and MS transfer line temperatures and F8 were prepared by dispersing xanthan gum in
were set at 240°C. The identification of the compounds purified water with continuous stirring.F6 was prepared
was based on the comparison of their relative retention by dispersing HPMCK15M powered in heated distilled
time and mass spectra with those from water (75°C) and the dispersion was cooled and left
(Wiley/ChemStation data system).12,13,29 overnight.20 In case of carbopol 940, triethanol amine
should added drop by drop to adjust the pH of the gel.21
Determination of Antimicrobial Activity of Myrtle Oil
The oil and aqueous phases of o\w emulsion were
Microbial Strains
prepared separately as follows, the oil phase was
The essential oil was tested against four clinical isolated prepared by mixing myrtle oil with olive oil with certain
strains provided by the Laboratory of clinical Laboratory amount of span 80, whereas the aqueous phase was
sciences department, Al mustansiriah Pharmacy College. prepared by dissolving the needed quantity of tween80 in
The identity of the microorganisms used in this study the Purified water, the quantity of surfactants used
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according to HLB theory. Methyl paraben and propyl effects like alteration in color, alteration in skin
paraben (preservatives) were dissolved in propylene morphology should be checked after 24 hrs then the
glycol and mixed with aqueous phase, then Sodium meta procedure repeated after one weak to ensure that there
bisulfate (antioxidant) added to the aqueous phase. is no sensitivity to the emulgel formula.17,32
Oily phase was heated to 70±1 °C. At the same time, Viscosity Study
aqueous phase was heated to 75±1 °C.
The viscosity of the selected formula F5 was carried out
Next, the aqueous phase was added to the oil phase in a with Brookfield Digital viscometer (LV DVE model) using
drop-to-drop basis with continuous stirring until cooled to S-64 spindle number.
room temperature.
100gm of the sample was placed in a glass container and
Finally Incorporation of emulsion into gel base by mixing the viscosity measured at different rates 2, 2.5, 5, 10, 12,
the both gel and emulsion in 1:1 ratio with continuous 20, 30, 50, 100 rpm, the temperature was maintained at
stirring.16 37° C. The viscosity was read directly after 30 seconds.34
Characterization and Evaluation of Myrtle oil Emulgel In vitro Antimicrobial Activity
Formulas
The optimum selected formula was tested for its
Physical Appearance antimicrobial activities.
All the prepared emulgel formulations were inspected The disc diffusion method was used for antimicrobial
visually for their color, homogeneity, consistency, screening as follows: A Sterile Mueller Hinton agar
grittiness and phase separation.24 medium was used for the antimicrobial assay of
Staphylococcus aureus, Pseudomonas aeruginosa.,
pH Determination
Streptococcus pyogen, and Sabouraud agar for the C.
The pH of all emulgel formulations was determined using albicans.
pH-meter by putting the tip of the electrode into the
The media were prepared and allowed to solidify in plates
emulgel without dilution and after (2 min) the result was 6
and then 0.1 mL of the microbial suspension (10 CFU/mL)
recorded.2,23
was streaked over the surface of the medium using a
In vitro Dissolution Test of Myrtle Oil Emulgel sterile glass spreader.
The in vitro release of myrtle oil from emulgel formulas The well were made and the size used was 6mm under
was performed by modified method using dissolution aseptic conditions and then sufficient amount of the
apparatus (paddle type) and dialysis membrane (M.WT formula was added into the pore.
12000).
The plates were then incubated for 24- 48 hours at 37°C
The membrane was soaked in phosphate citrate buffer of in order to get reliable microbial growth. Microbial
pH 5.5 for 24 hours and opened from both sides and then inhibition zones were measured measured in millimeters
one of the parties was tightly closed by elastic rubber. using a ruler. All tests were performed in triplicate.26
A 2 gm of emulgel (that contain 80mg of myrtle oil) was RESULTS AND DISCUSSION
placed inside the membrane and then the free end of
Identification of Myrtle Oil Components
membrane was closed by another rubber.
Extraction of the essential oil from the Myrtus communis
The membrane was fixed around the paddle of the USP
L. leaves produced a light yellow essential oil at 0.55 %
dissolution test apparatus and immersed in the
yield. The components of myrtle oil can be identified by
dissolution jar (previously filled with 500ml of phosphate
The GC/MS instrument. Approximately 19 compounds,
citrate buffer (pH 5.5) at 32±0.5°C with stirring rate of 50
representing 99.487% area of the essential oil, were
rpm.25,33
identified by GC-MS analysis of myrtle oil revealed that
Samples of 5 ml were taken at intervals of 30, 60, 90, 120, the major constituents of the oil were: alpha Pinene
150, 180, 210, 240, 270, 300, 330 and 360 min. (32.65.1%), 1,8Cineole (25.1%), Linalool (16.37%),
Eugenol (6.56%). The components of myrtle oil were
The samples were filtered through a filter (0.45 μm,
illustrated in Table 2.
millipore) and analyzed at λmax of myrtle oil using UV-
visible spectrophotometer. Antimicrobial activity of myrtle oil
The drug release experiments were conducted in In vitro antibacterial activity of M. communis EO was
triplicate (n = 3). assessed by the disc diffusion method against three
strains of bacteria: Staphylococcus aureus, Streptococcus
Skin Irritation Study
pyogen, Pseudomonas aeroginosa and against candida
After selection the best formula which gave the highest albican was expressed as diameter of the inhibition zones
percentage of release. The selected formula was applied Table 3. Myrtle oil has a good antibacterial and antifungal
on the properly shaven skin of three rats and its adverse activities as documented in many published papers which
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Int. J. Pharm. Sci. Rev. Res., 40(2), September – October 2016; Article No. 50, Pages: 271-277 ISSN 0976 – 044X

conclude that the antimicrobial activity of myrtle oil is It was obtained that liquid paraffin emulgel prepared
related to its components, each one of these components formulas were generally white creamy appearance with
has a specific mechanism contribute to its antimicrobial good consistency while olive oil emulgel prepared
activity.18,27 formulas has light yellow appearance with smooth and
excellent homogenous consistency. Carbopol 940 based
Determination Myrtle Oil λ max
emulgel formulas gave formulations very thick than other
The λ max of myrtle oil in phosphate citrate buffer formulas.
solution (pH5.5) and in absolute ethanol was 280
35 pH Determination
nmWhich is closely related to the λ max of Eugenol.
The pH values of all prepared formulas ranged from 5.17
Characterization of Myrtle Oil Emulgel Formulas
to 6.62 and this matched the requirements for topical
Physical Appearance preparations to avoid skin irritation.
Table 1: Main Compositions of Myrtle Oil Emulgel Formulas
Emulgel formulas

Propylene glycol
Methyl cellulose

Methyl paraben

Propyl paraben
Xanthan gum
Carbopol 940

Sodium meta
HPMCK15M

bisulphate
Myrtle oil

Tween 80
gumgum

Olive oil
Span 80

Water
F1 4 3.5 5 0.5 1.5 8 0.03 0.01 0.1 100
F2 4 3.5 5 1 3 8 0.03 0.01 0.1 100
F3 4 1 5 1 3 8 0.03 0.01 0.1 100
F4 4 1 5 0.5 1 8 0.03 0.01 0.1 100
F5 4 1 5 1 3 8 0.03 0.01 0.1 100
F6 4 2 5 1 3 8 0.03 0,01 0,1 100
F7 4 2 5 1 3 8 0.03 0.01 0.1 100
F8 4 1 5 1 3 16 0.03 0.01 0.1 100

Table 2: The Components of Myrtle Oil.

Peak Ret Index R. time Components Area %
1 920 4.892 Butyl isobutyrate 1.83
2 948 5.407 alpha-Pinene 32.65
3 943 6.050 beta-Pinene 0.45
4 958 6.227 beta.-Myrcene 0.38
5 969 6.508 alpha.-Phellandrene 0.12
6 919 6.629 4-Carene 0.20
7 1059 7.017 Cineole 25.1
8 976 7.223 Ocimene 0.33
9 1018 7.442 D-Limonene 0.47
10 1052 7.955 Terpinolene 0.51
11 1082 8.183 Linalool 16.37
12 1123 8.3 cis-Linaloloxide 0.35
13 1392 8.66 Eugenol 6.56
14 1143 10.188 alpha.-Terpineol 0.21
15 1270 10.583 Lavandulol 5.09
16 1386 11.586 Aromadendrene 1.90
17 1352 12.042 Geraniol acetate 2.73
18 1579 12.136 alpha.-Humulene 2.14
19 1507 12.411 Caryophyllene oxide 2.08

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Table 3: Antibacterial inhibition Zone Diameter of Myrtle Oil Including the Size of the Borer

Zone of Inhibition (mm)

Sample Conc.
Code mg\ml Pseudomonas
Staphylococcus aureus Streptococcus pyogen Candida albican
aeruginosa
1 500 31mm ± 0.6 29mm ± 1.6 28mm ± 0.6 47mm ± 1.8
2 250 29mm ± 0.9 27mm ± 1.9 26mm ± 0.7 36 mm ± 1.4
3 125 27mm ± 1.2 25mm ± 1.5 23mm ± 0.9 31 mm ± 1.9
4 62.5 24mm ± 1 22mm ± 2 21mm ± 0.4 25mm ± 1.5
5 31.25 21mm ± 0.9 17mm ± 1.6 _ 22mm ± 1.2
6 15.625 16mm ± 0.5 _ _ *
DMSO _ _ _ _

(values are mean ± SD) (n=3); _ mean no inhibition; * Mean not tested; All the result including the diameter of the disc
Table 4: Antimicrobial Inhibition Zone Diameter of F5

Staphylococcu aureus Streptococcus pyogen Pseudomonas aeruginosa Candida albican

F5 21mm ±1.4 20mm ± 2 17mm ±1.7 25mm ± 0.9

C - - - -

(Values are mean ±SD) (n=3); C (negative control) =Formula (F5) without myrtle oil; (-) Mean no inhibition zone.
All the result including the diameter of the disc (6mm)

Figure 3: Photographs of zones of inhibition of F18 against (A) Staphylococcus aureus, (B) Streptococcus pyogen, (C)
Pseudomonas aeruginosa, (D) Candida albican

In vitro Dissolution Test of Myrtle Oil Emulgel as shown in Figure 1, these results may be explained
according to the concept of escaping tendency of drugs,
Effect of Concentration of the Oil used in the Preparation
i.e., that increase the concentration of the oil result in
of the Emulgel
increased the entrapment of the drug within the formula
It was observed that by increasing the concentration of with subsequent reduction in drug release rate and
22,28
olive oil from 8% in F5 to 16% in F8 resulted in significant extent.
decrease in the release of myrtle oil after 6 hrs (p < 0.05)

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Effect of the Type of Gelling Agents with each of the generated as shown in Figure 2.The rheological behavior
Utilized Oil of formula (5) indicated that the systems were non-
newtonian in nature showing shear thinning and decrease
The effect of the gelling agents on the release of myrtle
in the viscosity with increasing speed rates. This pseudo
oil was shown in the Figures 1. It was observed that there
plastic rheology behavior of formulation is useful for
was a significant increase (p<0.05) in the amount of
topical application of emulgels.
Myrtle oil released after 6 hrs. from F5 as compared with
F2, F6 and F3.The order of the release in the olive oil
formulas was F5 (97.3±0.3%) > F2 (85.1±0.2%) > F6
(78.6±0.1%) >F3 (46.9±0.5%).
Effect of Concentration of Gelling Agent
F5 and F7 were exploited to study the significance of
increasing the amount of xanthan gum, in which 1% and
2% were used respectively. The result showed a
significant (p<0.05) decreasing in the release of Myrtle oil
due to increasing the viscosity of the formula with
increasing the amount of xanthan gum as shown in Figure
1.
Effect of the Total Amount of Surfactant Figure 2: Rheological Profile of Formula 18.
The effect of increasing the concentration of surfactants In vitro Anti-Bacterial and Antifungal Activity
(span80 and tween80) from 2%in (F4) to4% in (F5) lead to Formula (5) exhibited good antibacterial activity against
significant increasing (p<0.05) in the amount of myrtle oil Staphylococcus aureus, Streptococcus pyogen and
released after 6hrs as shown in Figure 1. This effect may Pseudomonas aeruginosa.and good antifungal activity
be referred to the ability of these emulsifying agents to against Candida albican, the diameter of inhibition zones
lower the interfacial tension between oil and aqueous excluding the size of the borer are illustrated in Table (4).
layer in the dispersion medium indicating an increasing
the hydrophilicity of emulgel which in turn increase Figure 3(A, B, C, D) show the inhibition zones of F5 against
penetration of dissolution medium into the emulgel the previous mention bacteria and fungi respectively.
structure and then increasing the amount of myrtle oil CONCLUSION
released.22
Herbal emulgel formulations containing myrtle oil were
prepared by two steps: emulsion preparation and then
incorporation into gel.
Emulgel Formulas containing myrtle oil were successfully
prepared with good physical properties, composed of
olive oil as oil phase with (span 80 and tween 80) as
emulsifying agents in addition to the gel phase which is
composed of either Methyl cellulose or Carbpol 940 or
Xanthan gum or HPMC as gelling agent.
The emulgel formula (F5), which has xanthan gum as a
gelling agent was chosen as an optimized formula among
all of the prepared formulas due to the excellent
Figure 1: Release Profile of Myrtle Oil from the Formulas consistency, homogenecity, spreading properties and the
highest percentage of drug release after six hours.
Skin Irritation Study
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