Development and Screening of Cinnamon Oil Based Topical

Formulations for their Antibacterial Activity

Tee Lui Shi

Bachelor of Pharmacy
School of Pharmacy
Taylors University

Supervised by
Dr. Manimaran Sellappan

Date

1.0 Introduction
Essential oils are aromatic, volatile mixtures of plant secondary metabolites, which cover
a broad spectrum of activities thanks to their chemical diversity- its common to find 20
60 constituents in an individual essential oil (Kalemba & Kunicka, 2003). Cinnamon oil
is one of those essential oils that have received much attention. For thousands of years,
cinnamon oil has been used as a spice due to its special aroma and now it has moved
beyond the realm of taste as more and more studies demonstrate the medicinal properties
of cinnamon oil (Bandara, Uluwaduge, & Jansz, 2012; Ranasinghe et al., 2013).
Although there are about 300 species of Cinnamomum, only four species are used to
obtain the spice cinnamon with Ceylon True cinnamon (Cinnamomum zeylanicum)
and Chinese Cassia cinnamon (Cinnamomum aromaticum) as the most widely available
varieties (Jayaprakasha & Rao, 2011). The purpose of present study is to develop
an effective topical antibacterial formulation of C. zeylanicum oil and to evaluate its
antibacterial activity. The cream formulated will be the first cinnamon oil antibacterial
cream targeted for human use. The rationale of choosing C. zeylanicum instead of C.
aromaticum is because the antimicrobial activities of C. zeylanicum oil are well
established and documented in the World Health Organization monograph (Who, 1999)
compare to the corresponding scanty information for C. cassia (Ooi et al., 2006).
2.0 Literature review
2.1 Phytochemical review
The plant organs of C. zeylanicum produce essential oil of varying chemical composition
with the major constituents of its leaf being eugenol (76.74-90.2%) (Jantan, Karim
Moharam, Santhanam, & Jamal, 2008; Paranagama et al., 2001; Singh, Maurya,
deLampasona, & Catalan, 2007).
For its bark, cinnamaldehyde (49.9-62.8%) is being reported as the primary constituent
(Paranagama et al., 2001; Jantan, Karim Moharam, Santhanam, & Jamal, 2008; Simi et
al., 2004; Singh, Maurya, deLampasona, & Catalan, 2007; Unlu, Ergene, Unlu,
Zeytinoglu, & Vural, 2010)
Camphor (47.42%) is being reported as the major constituent of C. zeylanicum root
(Paranagama et al., 2001).
Cinnamaldehyde and eugenol have also been reported as the major components of
Cinnamomum spp. essential oil extract (Azeredo, Santos, Helena, Noronha, & Maia,
2014; Cheng, Liu, Hsui, & Chang, 2006; Usta, Kreydiyyeh, Barnabe, Bou-Moughlabay,
& Nakkashchmaisse, 2003).

This chemical diversity of C. zeylanicum has been accounted for its wide variety of
medicinal benefits and usage in different industries (Ranasinghe et al., 2013).
2.2 Biological review
Besides its traditional use as a spice in flavouring industry and as a rub to increase blood
circulation in aromatherapy (Rana, Singh, & Gwal, 2011), modern studies from different
parts of the world have demonstrate numerous beneficial health effects of C. zeylanicum,
such as anti-inflammatory (Vetal, Bodhankar, Mohan, & Thakurdesai, 2013), antioxidant (Jayaprakasha & Rao, 2011; Singh et al., 2007) and antimicrobial (Naveed et al.,
2013; Ooi et al., 2006)
Anti-bacterial action of cinnamon oil is considered to arise mainly from the potential of
hydrophobic essential oil to disrupt the bacterial cell membrane and its structures which
leads to ion leakage (Bakkali, Averbeck, Averbeck, & Idaomar, 2008; Burt, 2004; Rana
et al., 2011).
Many studies have shown that cinnamaldehyde is the primary compound responsible for
major antibacterial activity of cinnamon oil (Rana et al., 2011; Unlu et al., 2010)
Cinnamon oil alone or in combination with triclosan, gentamicin, or chlorhexidine can
effectively inhibit biofilm formation, detach existing biofilms, and kill bacteria in
biofilms of clinical Staphylococcus epidermidis strains without causing resistance
(Hamidpour, Hamidpour, Hamidpour, & Shahlari, 2015).
Cinnamon oil together with its main constituents- cinnamaldehyde (0.05%v/v) and
eugenol (0.01%v/v) is reported to be effective against Pseudomonas aeruginosa and
Escherichia coli as they can markedly inhibit the biofilm formation of these bacteria by
96% and 90% respectively (Kim, Lee, Kim, Baek, & Lee, 2015).
Study done using disc diffusion method showed that essential oil of cinnamon fully
inhibit the growth of four gram-positive and gram-negative bacterial strains, namely
Pasturella multocida, Escherichia coli, Bacillus subtilis and Staphylococcus aureus
(Saleem, Bhatti, Jilani, & Hanif, 2015).
In a study done on cinnamon, geranium and lavender essential oils, cinnamon bark oil
was the most active against clinical and environmental strains of Acinetobacter
baumannii with MIC values ranging from 0.5 to 2.5 L/mL (Sienkiewicz et al., 2014).
In a study done on essential oils of cinnamon, rosemary, thyme, clove and mint,
cinnamon oil showed the strongest bactericidal effect against Escherichia coli (Kalaba &
Kalaba, 2014).

Essential oil obtained from C. zeylanicum inhibited activity of Staphylococcus,

Enterococcus, Enterobacter and Acinetobacter genera even in low concentration, with
the MIC for Gram-positive bacteria between 01.25-1.5 L/mL and for Gram-negative
between 1.0-1.75 L/mL. No sign of resistance as those seen with the recommended
conventional antibiotics was observed (Urbaniak, Gowacka, Kowalczyk, Lysakowska,
& Sienkiewicz, 2014).
Among all four selected essential oils, oil from the bark of C. zeylanicum showed best
antibacterial activity against all tested multidrug-resistance strains in the MIC assay,
namely, Salmonella typhi (D1 Vi-positive), Salmonella typhi (G7 Vi-negative),
Salmonella paratyphi, Escherichia coli, Staphylococcus aureus, Pseudomonas
fluorescens and Bacillus licheniform with the most prominent ones being 2.9 mg/mL
concentration against S typhi G7 Vi-negative and P fluorescens strains (Naveed et al.,
2013).
C. zeylanicum essential oil suppressed the growth of Acinetobacter spp. and exerted
synergistic effect with the antibiotic amikacin by drastically reducing the MIC of
amikacin when its sub-inhibitory concentrations at 78.125g/mL (MIC/8) is added to
the growth medium (Guerra et al., 2012).
In a study involving passaging bacteria up to fifty times with sequential exposure of
cinnamon essential oils, no resistance was reported out of the 48 clinical isolates and 12
reference strains under study (Becerril, Nern, & Gmez-Lus, 2012).
C. zeylanicum showed the highest activity among 19 essential oils when screened against
four bacteria strains namely Pseudomonas aeruginosa, Staphylococcus aureus,
Salmonella typhimurium and Bacillus subtilis, with Staphylococcus aureus and B.
subtilis being most susceptible, exhibiting MIC values 0.2 and 0.4 L oil/well
respectively (Rana et al., 2011).
Commercially available sugar-sweetened cinnamon chewing gum may benefit halitosis
by reducing volatile sulfur compounds producing anaerobes in the oral cavity as there
was significant reductions in total salivary anaerobes (p < 0.01) and H2S-producing
salivary anaerobes (p < 0.01) after subjects chewed gum containing cinnamic aldehyde
and natural flavors (Zhu, Carvalho, Scher, & Wu, 2011).
Among 21 essential oils tested, cinnamon oil was the most effective antibacterial agent
with MIC values ranging from 0.8 to 3.2 mg/mL to show maximum activity against P.
aeruginosa, B. subtilis, P. vulgaris, K. pneumonia and S. aureus (Prabuseenivasan,
Jayakumar, & Ignacimuthu, 2006).

Potential use of Cinnamomum zeylanicum bark oil in the formula of natural remedies for
the topical treatment of infections is demonstrated in a study by using disc diffusion
(DD) and minimum inhibitory concentration (MIC) methods. The essential oil showed
strong antibacterial activity against all 21 bacteria species tested and was highly effective
against Gram positive Staphylococcus, Streptococcus, Enterococus and Gram negative
Pseudomonas aeruginosa with a MIC of <0.04-1.12mg/ml to produce zone of inhibition
of 18- >40mm(Unlu et al., 2010).
Cinnamomum zeylanicum essential oils inhibited growth of Salmonella typhi,
Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Bacillus subtilis
with gram-negative organisms being more susceptible than gram-positive ones (Dubey,
Rana, & Shukla, 2005).
Essential oil obtained from the stem-bark of Cinnamomum species exhibit significant
antibacterial activity against a wide range of Gram positive and Gram negative bacteria,
especially Methicillin-resistant Staphylococcus aureus (MRSA) with zone of inhibition
up to 21.0000 1.4142mm (Buru, Pichika, Neela, & Mohandas, 2014) *not
c.zeylanicum, but got cinnamaldehyde*
3.0 Objective

To develop a new antibacterial topical formulation for human use using C. zeylanicum
essential oils.

To provide an alternative for the treatment of infections caused by resistant and

multi-resistant bacteria.

4.0 Scope
The rising of antibiotic resistant bacteria in the recent decades has become a serious threat to
public health worldwide (Handali, Hosseini, Ameri, & Moghimipour, 2011; Warnke et al.,
2009). This resistance towards antibiotic is fuelled by its widespread misuse, leading to
insufficiency of antibiotic therapies that are effective. There is a strong need for the
development of new antibiotic, however, designing and developing new drugs takes a lot of
time and money. This has caused many major pharmaceutical companies to abandon the field,
especially when the recuperation rate of R&D expenses are low due to rapid emergence of
bacterial resistance shortly after the drug is marketed. If this crisis situation persists, experts
predict that a pre-antibiotic era will return, where patients may routinely die from seemingly

minor infections (Qureshi, Agrawal, Madan, Pandey, & Chauhan, 2015). Thus, a different
alternative should be explored, that being the essential oils which affect a wide range of
antibiotic resistant microbes.
5.0 Research Methodology
5.1 Collection and identification of essential oils
The essential oil of C. zeylanicum is to be purchased from Malaysian supplier in May 2015.
Identity and quality of the essential oil is verified through the specifications in certificates of
analysis provided by the suppliers.
5.2 Development of formulations
This product will be the first antibacterial cinnamon oil cream that is targeted for human use.
Cream formulation is chosen as topical formulation is said to be advantageous in the aspects
of solubility, concentration of drug released and delivery of drug directly to the site of action
(Okore, Attama, Ofokansi, Esimone, & Onuigbo, 2011; Woodruff J, 1995). On the other
hand, cream formulation is easily spreadable and washable with water which facilitate its
application (Muller et al., 2003; Okore et al., 2011; Orafidiya, Oyedele, Shittu, & Elujoba,
2001; Sellappan M., 2014). The base cream contains water and oil phases, suitable excipients
are added to the formulations besides the cinnamon oil. The tentative formula of the cream is
illustrated in the tables below.
Table 1: Tentative formula of the 2% cream formulation (100g)
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

Ingredient
Stearic acid
Potassium hydroxide
Cetosteryl alcohol
Liquid paraffin
Petroleum jelly
Methyl paraben sodium
Propyl paraben sodium
Glycerin
Cinnamon oil
Purified water

% w/w
16.00
02.00
05.00
03.50
03.50
00.16
00.04
07.00
02.00
60.80

Table 2: Tentative formula of the 5% cream formulation (100g)

1.
2.
3.
4.
5.

Ingredient
Stearic acid
Potassium hydroxide
Cetosteryl alcohol
Liquid paraffin
Petroleum jelly

% w/w
16.00
02.00
05.00
03.50
03.50

6.
7.
8.
9.
10.

Methyl paraben sodium

Propyl paraben sodium
Glycerin
Cinnamon oil
Purified water

00.16
00.04
07.00
05.00
57.80

Table 2: Tentative formula of the 10% cream formulation (100g)

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

Ingredient
Stearic acid
Potassium hydroxide
Cetosteryl alcohol
Liquid paraffin
Petroleum jelly
Methyl paraben sodium
Propyl paraben sodium
Glycerin
Cinnamon oil
Purified water

% w/w
16.00
02.00
05.00
03.50
03.50
00.16
00.04
07.00
10.00
52.80

5.3 Procedure
Aqueous cream seems to be as effective as the test agents because of its easy penetration
through the skin (Handali et al., 2011; Muller et al., 2003; Orafidiya et al., 2001). Stearic
acid, cetosteryl alcohol, liquid paraffin and petroleum jelly to be melted on a steam bath at
75oC. The remaining ingredients are to be dissolved in water and boiled at 75 oC. This
aqueous solution to be added to the above oily phase with agitation. Potassium hydroxide
reacts with a portion of stearic acid to form potassium stearate, which emulsifies the
unreacted stearic acid as dispersed phase. Excessive agitation to be avoided so as to avoid air
getting entrapped within the cream. 2% and 5% of the essential oils are to be mixed
uniformly with the above prepared cream base individually and combination of both the oils
with equal proportions using a mechanical stirrer. Glycerin to be added finally and mixed.
The formulated creams are filled in a suitable plastic container.
5.4 Evaluation of formulations
The prepared cream formulations are to be subjected to various physical evaluations listed
below (Baboota et al., 2011; Okore et al., 2011).
5.4.1 Physical Evaluation
A) Physical appearance

The formulated creams are observed visually for their visual appearance, transparency,
colour, consistency and feel upon application such as stickiness, smoothness and cooling
effects.
B) Determination of pH
The pH of the prepared cream formulations and the base are to be determined using a pH
meter (Eutech cyber scan pH 310) by preparing 1 gm of cream formulation to dissolve in
100 ml of water and maintaining a temperature of 25oC. The averages of the triplicate
observations will be recorded.
C) Determination of Extrudability
Extrudability is a useful empirical test to measure the force required to extrude the material
from a tube. Since packaging in collapsible tubes has gained considerable importance in
delivery of the desired quantity of cream, measurement of extrudability becomes an
important critical data for creams. In the present study, the method adopted for evaluating
cream formulation for extrudability will be based upon the quantity (in percentage) of the
cream extruded from a collapsible tube on application of certain load. The more the quantity
extruded, the better is the extrudability.
The cream formulation to be filled in a standard capped collapsible sealed tube. The tube will
be weighed and recorded. The tube will be placed between two-glass slides and is clamped. A
500 g weight to be placed over the glass slide and then the cap is opened. The amount of
cream extruded is collected and weighed. The percentage of cream extruded will be
calculated and recorded as per grades allotted.
D) Determination of Viscosity:
The viscosity of formulated cream bases was determined. The viscosity determinations were
carried out on Brookfield viscometer using spindle number S06 and the determinations
were carried out in triplicate and the average of three reading is recorded.
Antibacterial assay
The cylinder plate assay of drug potency is based on the measurement of the diameter of
zones of microbial growth inhibition surrounding the cylinder (cups) containing various
dilutions of test compounds. The following Gram-positive and Gram-negative bacteria will

be used for the

present study to determine the antimicrobial activity (Manimaran,

Themozhil, Nanjan, & Suresh, 2007) (Baker and Breach, 1980; James et al., 1992).
5.5 Procedure
Petri dishes are to be cleaned, dried and sterilized. Then they are filled with a Nutrient
Agar or SDA medium with uniform thickness. After solidifying, the plates are to be
inoculated with the above mentioned organisms of Gram positive, Gram negative
bacteria and fungi. In the first plate, five holes to be made in the inoculated plates by
means of aluminum or stainless steel sterile borer with a height of 10 cm and an internal
diameter of 6-8 mm. To the first, second and third hole, required quantity of 2% cream
of oregano oil, geranium oil and mixed cream formulations to be introduced. The
standard drug and cream base to be filled in the fourth and fifth hole. The same
procedure will be followed for 5% cream formulations. Then the plates are to be kept
in the refrigerator for 2 hrs for diffusion. All the plates are to be incubated at 37 oC for
24 hrs. All the standards to be inoculated separately for bacteria and fungi.. The zone
of inhibition was measured by using antibiotic zone reader.
6.0 Outcome
This research project focuses on the three Ps: people, planet and profits. Thus, its
outcome can be simplified as triple bottom. The outcomes are categorized into direct
outcomes and indirect outcomes.
6.1 Direct outcome

A new antibacterial formulation consisting of cinnamon oil will be developed. There

is currently no product for human use being marketed which uses cinnamon oil as the
main active ingredient into a formulation.

There will be more alternatives to treat an infection caused by MRSA strains.

6.2 Indirect outcome

It encourages continuous research and development in the field of essential oils.

Potential anticancer, antidiabetic, anti-inflammatory and other drugs can be
developed.

7.0 Timeframe
Duration
Activity

Apr

May

June

2014
July Aug

Sept

Oct

Nov

Literature search

Proposal writing

Proposal presentation

Collection of materials

Formulation development

Physical & biological

evaluation of the

formulation
Data analysis

Thesis writing

Thesis presentation

8.0 Budget
No

Description

Quantity

(kg)
1. Stearic acid
0.100
2. Potassium hydroxide
0.100
3. Cetosteryl alcohol
0.500
4. Liquid paraffin
0.500
5. Petroleum jelly
0.500
6. Methyl paraben sodium
0.100
7. Propyl paraben sodium
0.050
8. Glycerin
\
9. Cinnamon oil
50ml
10. Nutrient agar- (to prepare 0.750
11.

media)
Sabourad dextrose agar (to
prepare media)

required Cost (RM)

50
550
100
300
1200
200
400
200
3400

They can contain up to 2060 constituents at various concentrations, but usually only 23
main constituents that are responsible for their aromatic and biological properties (Kalemba
& Kunicka, 2003).

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Becerril, R., Nern, C., & Gmez-Lus, R. (2012). Evaluation of bacterial resistance to
essential oils and antibiotics after exposure to oregano and cinnamon essential oils.
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Singh, G., Maurya, S., deLampasona, M. P., & Catalan, C. a N. (2007). A comparison of
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Cinnamomum zeylanicum Blume, Lauraceae, has many biological properties as analgesic,

antiseptic, antispasmodic, aphrodisiac, astringent, carminative, haemostatic, insecticidal and
parasiticide. Barks from branches, without the epidermis and subereous layer, is marketed as
the commercial cinnamon which has long use in perfumery, culinary and native medicine
fields (2,20). Previous research has revealed interesting antimicrobial effect in C.
zeylanicum essential oil (10,27,28). Camphene, linalool, a-phelendrene, a-terpinene,
limonene, b-cymene, a-cariophyllene, cinnamaldheyde and eugenol are some of the
compounds found in C. zeylanicumessential oil (24,44).

The present study aimed to evaluate the effect of C. zeylanicum Blume essential oil on the
growth and morphogenesis of some Aspergillus species known as potential etiological agent
of fungal infections.
http://www.dermnetnz.org/bacterial/
Skin bacteria
Staphylococcus aureus
Folliculitis
Furunculosis (boils) and abscesses
Impetigo (school sores)
Methicillin (meticillin) resistant Staph. aureus
Staphylococcal scalded skin syndrome
Toxic shock syndrome
Tropical pyomyositis
Botryomycosis (pyoderma vegetans)
Streptococcus pyogenes
Cellulitis
Erysipelas
Impetigo
Necrotising fasciitis
Infectious gangrene
Scarlet fever
Rheumatic fever, erythema marginatum
Overgrowth of Corynebacterium spp. (erythrasma, pitted keratolysis & trichomycosis
axillaris)
Less commonly, other bacteria may also cause infection with skin signs. These include:
Neisseria species, cause of gonorrhoea and meningococcal disease
Erysipelothrix insidiosa, cause of erysipeloid (usually an animal infection)
Haemophilus species, cause of chancroid and cellulitis in young children
Helicobacter pylori, a stomach infection, which may be associated with some cases of
chronic urticaria and rosacea
Klebsiella rhinoscleromatis, cause of rhinoscleroma
Mycoplasma pneumoniae, a cause of pneumonia, causes non-specific erythema,
bullous eruptions, urticarial rashes, erythema multiforme, mucositis and
rarely, SJS/TEN
Pseudomonas aeruginosa causes wound infections, athlete's foot, gram negative
folliculitis, chronic paronychia (green nail syndrome), spa pool
folliculitis and ecthyma gangrenosum
Calymmatobacterium granulomatis, cause of granuloma inguinale
Bacillus anthracis, cause of anthrax
Clostridium perfringens and other species cause gas gangrene
Treponema species cause syphilis, yaws and pinta
Bartonella species cause cat scratch fever, bacillary angiomatosis and bartonellosis
Mycobacterium species cause tuberculosis, leprosy and atypical mycobacterial
infections including Buruli ulcer
Leptospira, cause of leptospirosis, which may cause bleeding into the skin (purpura)
Nocardia, cause of nocardiosis

Yersinia pestis, cause of bubonic plague, which causes swollen lymph glands and
pustules, ulcers and scabs on the skin
Serratia marcescens, a facultative anaerobic gram-negative bacillus that may rarely
cause skin infections such as cellulitis, abscesses and ulcers; usually in patients
with immunodeficiency.
Fusibacterium species, Bacillus fusiformis, Treponema vincenti and other bacteria
may result in tropical ulcer
Burkholderia species, cause of melioidosis and glanders, in which abscesses may be
associated with systemic symptoms.
Actinomcyes species, cause of actinomycosis, in which granular bacteriosis occurs i.e.
abscesses and sinus tracts draining sulphur-yellow granules.
Vibrio vulnificus, a cause of septic shock characterised by blood-filled blisters.
Brucella species, cause of brucellosis, a febrile illness caught from unvaccinated
animals or their unpasteurised milk.
Salmonella species, particularly S typhi (typhoid fever)
Aeromonas found in water rarely causes skin and soft tissue infections