Effects of Rosemary Leaves Extract (Rosmarinus Officinalis L.

)
on Angular Cheilitis Healing of Wistar Male Rat Induced by
Staphylococcus aureus and Candida albicans

Directed by:

Sumintarti
Nurul Fatimasari S.

Department of Oral Medicine,

Faculty of Dentistry,
Hasanuddin University,
Makassar, Indonesia
 Introduction

Indonesia is the second largest country in the world

after Brazil is rich in biodiversity with a variety of
plants that are available about 30,000 species,
including medicinal plants amounting to 2,500 species.

Various kinds of plants have great benefits in life. One

of the benefits is as an herbal medicine.
Global back to nature trends have
contributed to increase public awareness of
organic food production and healthier, safer,
and cheaper raw materials based on natural
ingredients.

One type of ornamental plant that has a dual

function as a medicinal ingredient is
rosemary known by the Latin name
Rosmarinus officinalis L.
Rosemary

Rosemary (Rosmarinus officinalis L.) is

native to the Mediterranean region and
Uruguay.

This specie grows worldwide and has been

cultivated since long ago, in ancient
Egypt, Mesopotamia, China and India.
 Scientific Classifications

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Lamiales

Family : Lamiaceae

Genus : Rosmarinus
Source: Umberto Quattrocchi. Crc
World Dictionary of Medicinal and
Poisonous Plants. Boca Raton: Species : Rosmarinus officinalis
Crc Press; 2012
Morfology of Rosemary
An erect that may
reach 2 m

It’s evergreen leaves are dark

green above and white hairy
below

The leaves are

2 to 3.5 cm in length

The flowers are small and colors

include purple, pink, pale blue
and white.
 Rosemary’s Constituents
Essential oils

• 0,5%-2,5% monoterpene with champhor (15%-25%),

cineol (15%-50%),α-pinene (10%-25%), camphene,
and borneol.

Flavonoids
• Diosmin, diometin, genkwanin, luteolin, and apigenin.

Phenolic Acids

• Rosmarinic acid and carnosic acid

Carnosol
• Phenolic diterpenes
 Beneficial Effects

Antibacterial Antifungal

Antiviral Antioxidant

Anti-
Anti-cancer
inflammatory

Anti-ulcer Anti-diabetic
 Rosemary in Household


Spices Decoration Eradicate Mosquitoes

Soap Shampoo and Lotion Aroma therapy

astringent, diuretik Infused Water
and tonic

Mouthwash and
Herbal medicine antiseptic of the mouth
 Angular Cheilitis

Angular Cheilitis is an inflammatory lesion

usually seen in labial commissures which is an
infections of Staphylococcus aureus and/or
Candida albicans at the corners of the mouth.

The lesion may be confined to the mucosa of

the lips but in some cases, the lesion may
extend to the vermilion border at the edge
corner of the lip attached to the skin.

Angular Cheilitis
Source: https://healthywildandfree.com/wp-
content/uploads/2017/03/Angular-cheilitis.jpg
 Clinical Symptoms

A roughly triangular area of erythema

Cracking

Fissures

Edema

Maceration at the corners of the mouth with

symptoms of soreness, pain, pruritus or burning or
raw feeling.
Etiology
Staphylococcus aureus infection

Candida albicans infection

Nutritional deficiencies such as iron

and vitamin B12

Improperly usage of dentures

causing decreased vertical
occlusal dimension.

Immunosuppression

Bad habits: wetting the lips with

saliva, licking the corners of the lips

Hypersalivation.
 Treatment

Candida albicans Staphyloccocus aureus

Antifungal ointment Antibacterial ointment

 anti-ulcer anti diabetic

anti-cancer antiviral

anti-
antifungal
inflammatory

Previous
research
antioxidant antibacterial
showed
rosemary
Especially against gram-
positive bacteria such as
Staphylococcus aureus and
Enterococcus faecalis as
well as on gram-negative
bacteria such as
Escherichia coli.
One of the infections in the oral
cavity caused by Staphylococcus
aureus is angular cheilitis.

Angular Cheilitis is an inflammatory

lesion usually seen in labial
commissures which is an infections
of Staphylococcus aureus and/or
Candida albicans at the corners of
the mouth.
Based on this explanation, the aim of this
study is to determine the effect of
rosemary leaves extract that used as an
alternative to the treatment of angular
cheilitis tested in Wistar male rat induced
by Staphylococcus aureus and Candida
albicans.
 Material and Methods

This research type was an experimental laboratory

with posttest only control group design.

This research was conducted at Phytochemistry

Laboratory and Biopharmaceutical Laboratory
Faculty of Pharmacy, Hasanuddin University.

This research was on May-June 2017.

The sample to be used in
this research was Wistar
The sampling technique
male rats (Rattus
used was simple random
novergicus) induced by
sampling.
Staphylococcus aureus
and Candida albicans.
 Staphylococcus aureus-
Group 1 induced and given
rosemary leaves extract

Staphylococcus aureus-
Group 2 induced and given
gentamicin
The subjects were 24
Wistar male rats divided
into four treatment groups
Candida albicans-
Group 3 induced and given
rosemary leaves extract

Candida albicans-
Group 4 induced and given
rosemary leaves extract
Rosemary leaves extract is a rosemary
plant which part of the leaves is
extracted with maseration method in
ethanol solvent with 70% concentration
then evaporated using rotary
evaporator to get viscous extract.

The selected
leaves are the old
ones which fresh,
dark green, and
the shape is still
intact.
The viscous extract was then
prepared in 100% concentration
by mixing NaCMC 1% solvent with
a ratio 1: 1 (the extract: solvent).

Inoculum Staphylococcus
aureus ATCC 25923 and
Candida albicans were
obtained from the
Microbiology Laboratory
Faculty of Pharmacy,
Hasanuddin University.
This research was done by making cuts
on both sides of the corner of the mouth
of Wistar male rats by mutually pulling
between the two corners of the upper
and lower lip together to make the
wound of the fissure

The wound is made of 4 mm so that

bacteria and fungi can invade quickly.

Induction was done with inoculum of

Staphylococcus aureus in the first and
second groups and inoculum of Candida
albicans in the third and fourth groups
using a sterile spherical ose.
the wound was left for 24 hours without being
treated to occur the growth of bacteria and
fungi so that the wound infection occured on
both sides corner’s mouth of Wistar male rats.

After 24 hours, the infected wound infections

were given rosemary leaves extract in the first
and third groups, gentamicin in the second
control group and miconazole in the fourth
control group applied topically.

The length of wound

and erythema were
observed of each
group for 7 days.
 Results

Based on table, all experimental animals from each group

treatment undergo a change that shows the length of
angular cheilitis wounds that are reduced day by day.
• Based on Graph 5.1, the curve appears to decrease in
all groups from day 0 to 7.

• The first group curve induced by Staphylococcus

aureus using rosemary leaves extract had an average
decrease in wound length greater than in the other
group, followed by a third group induced by Candida
albicans using rosemary leaves extract as a cure
agent.
• With the decrease, descriptively from rosemary leaves
extract when induced with Staphylococcus aureus
and Candida albicans, has an effect on the healing of
angular cheilitis.
 Friedman's non-parametric statistical test was
performed from the data obtained to determine the
differences in wound length of each treatment group on each
observation day. Since p = 0,000 <0,05, Ho was rejected.
This means that there is a significant difference in every
observation day of the first, second, third and fourth groups
 Based on table, the first treatment group induced by Staphylococcus aureus
using rosemary leaves extract showed that almost all treatment groups experienced
significantly reduced wavelength change, except on the fifth day to the sixth day and
the sixth day to the seventh day was not significant because p > 0.05.
 Based on table of the Wilcoxon test of the second treatment group induced by
Staphylococcus aureus using gentamicin as the ingredient for healing, it was seen that
all treatment groups experienced a significant change in reduced wavelength, except
on the sixth day to the seventh day seen as insignificant because p> 0.05.
 Based on table of the Wilcoxon test of the third treatment group induced by
Candida albicans using rosemary leaves extract as the ingredient for healing, it was seen
that all treatment groups experienced a significant change in the decrease in the length of the
wound, except on the sixth day to the seventh day was not significant because p> 0, 05.
 Based on table the Wilcoxon test of the fourth treatment group induced by
Candida albicans using miconazole as the ingredient for healing, showed that all
treatment groups experienced a significant increase in the decrease in the length of
the wound with a value of p <0.05.
• Based on Mann Whitney test results, the first and second
groups induced by Staphylococcus aureus using rosemary
leaves extract as a healing agent and using gentamicin as
control, obtained a significance value of p <0.05 on the
second, third and fourth day, meaning that on the second,
third day , and fourth there was a significant difference
between rosemary leaves extract and gentamicin.

• Based on Mann Whitney test results of Candida albicans-

induced third and fourth groups using rosemary leaves
extract as a healing agent and using miconazole as its
control, obtained a significance value of p <0.05 on the first,
second, third and fourth day, meaning that on the first day ,
second, third, and fourth, there was a significant difference
between rosemary leaves extract and miconazole.
 Discussion
• Based on the results of this study, rosemary leaves extract
has an effect on the healing of angular cheilitis in Wistar
male rats induced by Staphylococcus aureus and
Candida albicans.

• The rosemary leaves contain essential oils, flavonoids,

phenolic acids, trycyclic diterpenes and ursolic acids
which potentially to be antibacterial, antifungal, antiviral,
antioxidant, anti-inflammatory, anticancer, anti-ulcer and
antidiabetic.

• Physical observation on the 0th day for all four groups

after treatment, bacterial infection occurred in the first
and second groups, which is indicated by tissue damage
in the area around the lesion.
• While in the third and fourth groups of fungal infections are
characterized by severe tissue damage in the area around the
lesion. Fungal infection wounds are more severe when compared
with bacterial infection wounds.

• On the first day the whole group undergoes an inflammatory phase

which is a live tissue defense reaction against all forms of wound
involving the functioning of blood and blood vessels, nerves,
lymph, fluids and cells around the wound.

• In the inflammatory reaction stage of infection occurs which is the

process of entry of a number of pathogenic microorganisms in this
case is Staphylococcus aureus and Candida albicans to the wound
area.

• A number of such pathogenic microorganisms will enter the tissues

through open areas due to injury.11
• According to Table 1, the first group induced by
Staphylococcus aureus using rosemary leaves extract
had a mean decrease in wound length greater than in
the other group, followed by group 3 induced by
Candida albicans, and rosemary leaves extract as a
healing agent.

• With the decrease, the descriptive rosemary leaves

extract has an effect on the healing of angular cheilitis.

• The rosemary leaves extract group has a better healing

effect than the control group of gentamycin and
miconazole which causes rosemary leaves have several
important components that help the healing process
such as essential oils, flavonoids, phenolic acids,
trycyclic diterpenes and ursolic acids that has a major
role as antibacterial, antifungal, antioxidant and anti-
inflammatory.
• The first group induced by Staphylococcus aureus and
given rosemary leaves extract were the faster group in
the treatment of angular cheilitis wounds compared
with other groups.

• This is in accordance with research conducted by

Moreno et al (2012) regarding rosemary extract as an
effective antibacterial against Gram-positive bacteria
common pathogenic bacteria such as Staphylococcus
aureus.

• Based on research conducted by Moreno et al (2012)

stated that rosemary leaf extract which has the content
of carnocic acid and carnosols that has a major role as
an effective antibacterial to Staphylococcus aureus.3
• Research conducted by Hernandez (2009) shows that
rosemary leaves contain high antioxidants due to the
presence of carnocic acid.12 Carnocic acid is one of the
rosemary content that has a major role as an antibacterial.13

• Research conducted by Moreno et al (2012) suggested that

the presence of intracellular antibacterial activity carnocic
acid against Staphylococcus aureus. Carnocic acid at
concentrations of Staphylococcus aureus has high selectivity
to bacteria and is non-toxic to macrophages.

• Another study of in vivo antibacterials from rosemary extract

conducted by Barni et al (2009) showed a large carnocic acid
content effect on Staphylococcus aureus in two trial skin
infections in mice.3
• Other major ingredients in rosemary leaves such as essential
oils, flavonoids and ursolic acid has an important role in
wound healing. Essential oils contain phenol and kavikol
which are useful as antimicrobial, antibacterial, and
disinfectant. The content can clean the wound and prevent
the occurrence of infection so it can accelerate the end of
the inflammatory phase in the wound healing process.14

• The content of rosemary leaves which also act as anti-

inflammatory and antioxidant is rosmarinic acid which is a
polyphenol compound. This was stated by a study conducted
by Joao Rocha et al (2014) that examined the anti-
inflammatory effect of rosmarinic acid and an extract of
rosemary in rat models of local and systemic inflammation.

• Based on these studies, rosmarinic acid showed good

anti-inflammatory activity, which includes its antioxidant
properties, inhibition of neutrophil activity, inhibition of
MMP-9 activity and modulation of the NF-jB.15
• According to Table 7, in the Staphylococcus aureus-induced
group, the first and second groups had significant differences
between rosemary leaves extract and gentamicin on the second,
third and fourth days.

• The effect difference between the first and second groups as

controls can be due to the active ingredient content contained in
the rosemary leaves that play an effective role as antibacterial
and anti-inflammatory while gentamicin only acts as an
antibacterial.

• While in Table 8, Candida albicans-induced group that is third and

fourth group there was a significant difference between rosemary
leaves extract and miconazole on the first, second, third and
fourth day.

• The effect difference between the third and fourth groups as

controls can be due to the active ingredients contained in the
rosemary leaves which also play an effective role as an antifungal
and anti-inflammatory. While miconazole only acts as an
antifungal.
• Based on the order of effectiveness of wound healing
process, the most effective rosemary leaves treatment
group was the induced group of Staphylococcus aureus
then Candida albicans while the most effective control
group was gentamicin and the last was miconazole.

• This happened because rosemary leaves extracts are

more effective against wound healing infections caused
by Staphylococcus aureus and infection wounds in the
group induced by Candida albicans resulting in more
severe injuries compared to the Staphylococcus aureus-
induced injury group.
 Conclusion

The rosemary leaves extract (Rosmarinus officinalis

L.) has an effect on wound healing of angular
cheilitis.

Rosemary leaves extract was more effective

against wound healing of angular cheilitis infections
Wistar male rats induced by Staphylococcus aureus
compared to angular cheilitis infections Wistar male
rats induced by Candida albicans.
THANK YOU
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