University of El Oued Chahid Hamma Lakhdar

University homepage: https://www.univ-eloued.dz/

studying the effect of siwak (salvadora persica) on the

microorganisms and gingivitis
, Ghania Nour Elyakine , Tedjini Farah Cheriet Soundes

Faculté des Sciences de la Nature et de la Vie, Département de La Biologie cellulaire

et Moléculaire, Spécialité: Biochimie Appliqué
Abstract

Objective: Periodontal disease is one of oral and dental diseases which most commonly found in humans
caused by several factors, one of them due to the accumulation of bacterial plaque. an antibacterial effect has
been postu- lated; however, tests of siwak extract from Salvadora persica (Arak) disclosed only low to
moderate antibacterial effects. This may be attributable to the extraction process. Our aim was to test in vitro
the antibacterial effect of miswak pieces, without extraction, on bacteria implicated in the etiology of
periodontitis and caries.

Material and Methods: siwak pieces were standardized by size and weight

(0.07 and 0.14 g) and tested against Streptococcus mutans, Lactobacillus acidophilus, Aggregatibacter
actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Porphyromonas gingivalis, and, as
a reference, Haemophilus

influenzae. The siwak pieces were tested in two ways: em- bedded in the agar plate or suspended above the
agar plate.

Results: The inhibitory effect was most pronounced on P. gingivalis,

A. actinomycetemcomitans, and H. influenzae, less on S. mutans, and least on L. acidophilus. Suspended miswak
had comparable or stronger effects than siwak em-

bedded in agar. The 0.14-g suspended siwak exhibited sig- nificantly greater inhibition on A.
actinomycetemcomitans and H. influenzae than the 0.14 g siwak embedded in agar (P <0.01 and P <0.001,
respectively).

Conclusions: siwak embedded in agar or suspended above the agar plate had antibacterial effects against all
bacteria tested. The antibacterial effect of suspended siwak pieces suggests the presence of volatile active
antibacterial compounds.

KEY WORDS

Aggregatibacter actinomycetemcomitans; antibacterial agent; chewing siwak; periodontitis; Porphyromonas

gingivalis; Streptococcus mutans; pellicle; extract
 Introduction
The oral macrobiotic are among the most
complex of the human body. More than
700 bacterial species have been detected
in the oral cavity, but more than half of
these species have yet to be cultured.
Dental plaque is a complex microbial
community, growing as a biofilm on tooth
surfaces. The etiology of dental caries and
various forms of periodontal disease has
long been recognized to be related to
bacterial accumulations and plaque
composition.
Dental plaque is a well-known etiologic
factor for gingival diseases.
Dental plaque is a biofilm defined as a
community of bacteria with extracellular
polymers attached to the surface.
In the oral cavity, the bacterial
community consists of various species It
is now widely
known that environmental changes such
as the use of orthodontic appliances have
the ability to change a bacterial
community, thereby leading to diseases
Dental plaque is solely responsible for
the initiation and progression of gingival
diseases Studies showed that strains of A.
actinomycetemcomitans and P. gingivalis
are capable of invading epithelial cells
derived from human epithelial
periodontal pockets or gingival sulci.
A. actinomycetemcomitans also produces
leukotoxin
that lyses polymorph nuclear leukocytes
and monocytes, which enables it to evade
the innate defense line
of the periodontal pocket P. gingivalis is
characterized by production of an
unusually extensive array of proteases,
collectively designated the gingipains.
These gingipains play major roles in P.
gingivalis colonization, host tissue
invasion, destruction of collagen and
fibrinogen, and hydrolysis of hemoglobin.
Good oral hygiene habits can prevent or
retard the development of caries,
gingivitis, and periodontitis. The main
tools for maintaining good oral hygiene
are
toothbrushes with tooth paste and
interdentally cleaners .Historically, the
first known oral hygiene tool is the
chewing
stick or siwak (also called siwak or
miswak).
The most common type of chewing stick,
siwak, is derived from Salvadora persica,
a small tree or shrub with a spongy stem
and root, which is easy to crush between
the teethSalvadorapersicaLinn. (siwak) is
an evergreen small tree that belongs to
the
Salvadoraceae family The S. persicatree
can reach up to three meters tall and has
thick succulent small leaves, new stem
branches are green to grayish in color
while old branches are dark brown . The
scientific name of S. persicawas given to
the tree after classifying the first sample
in 1598 by the Spanish botanist, Dr.
Laurent Garcin,
who collected the specimen from the
middle-east . The tree is globally known
as the toothbrush tree or chewing-sticks;
it has many local names in different
geographical regions such as: siwak or
Arak in Arab world, Koyoji in Japan,
Qesam in Hebrew, and
Mastic in Latin .
While siwak had been used by various
civilizations for the Arabs it was only
during the Islamic period that personal
hygiene was further emphasized as part
of religious obedience, including the use
of siwak as a tool for oral hygiene .As a
display of obedience to religious advice,
Today, the siwak practice continues and
is typically recognized as a cultural
identity among Muslim communities.
The World Health Organization
recommends and encourages the use of
chewing sticks as an effective tool for oral
hygiene in areas where such use is
customary.
Clinical studies comparing mechanical
plaque removal by the siwak and the
modern toothbrush reported that the
siwak is as good as or more effective than
tooth brushing in reducing plaque and
gingivitis.The use of siwak has also been
reported to inhibit the formation of
dental plaque chemically and exert
antimicrobial effect
against many oral bacteria
In addition to its mechanical effect, the
siwak exerts chemical activity against
bacteria and plaque formation. In vitro
studies showed that aqueous siwak
extract (prepared from Salvadora
persica) exerts antimicrobial activity
against some oral bacteria,
The World Health Organization (WHO)
recommends and advocate siwak as an
effective tool for oral hygiene, with
softwood fiber mechanical action and its
chemical therapeutic action. Siwak
contains essential oils and a variety of
other chemical compounds, i.e. Anorganic
compound, such triethylamine, alkaloids,
flavonoids, anthraquinone, tannins,
saponins, sterols, vitamin C, and
inorganic compounds, such chloride,
calcium,
a large amount of fluoride, silica and
sulfur chemical components of essential
oils contained in siwak leaves such benzyl
nitrile, eugenol, thymol, isothymol,
eucalyptol, isoterpinolen, and beta-
caryophyllenchemical .The research
results of inhibition test of siwak oral
bacteria showed that siwak wood have
inhibitory activity against oral bacteria.
Therefore, the authors are interested in
investigating the effectiveness of siwak
against the pathogenic bacteria causing
periodontal disease.
Aims
This study aims to systematically
review the literatures on the nature,
and extent of siwak use On the nature
of using the siwak and how it has an
anti-bacterial effect on oral bacteria
and gingival diseases.
the relation between Dental plaque
and oral bacteria
Dental plaque is a general term for the
complex microbial community that
develops on the tooth surface, embedded
in a matrix of polymers of bacterial and
salivary origin. Plaque is composed of
organic, inorganic materials derived from
saliva, gingival crevicular fluid et
bacterial
products. The organic constituents of
plaque include polysaccharides, proteins,
glycoproteins et lipid material. The
inorganic constituents of plaque include
primarily of calcium et phosphorus et
traces of sodium, potassium
The process of plaque formation can be
divided into three phases:
a) Formation of Dental Pellicle
b) Initial colonization of bacteria phosphoproteins and lipids, including
statherin, amylase, proline-rich
c) Secondary colonization & plaque
peptides (PRPs) .For example, cells of
maturation.
Actinomyces viscosus possess fibrous
What is Dental Pellicle? protein structures called Fimbriae that
extend from the bacterial cell surface.
Pellicle is a glycoprotein, derived from
Protein adhesins on these Fimbriae
components of saliva and crevicular fluid
specifically bind to proline-rich
as well as from bacteria and host tissue
proteins that are found in dental
cell products and debris. Pellicle is
pellicle, resulting in the attachment of
formed on all surfaces of the oral cavity,
the bacterial cell to the pellicle-coated
including all tissue surfaces as well as
tooth surface. The plaque mass then
surfaces of teeth and fixed and removable
matures through the growth of
restorations if any. Pellicle functions as a
attached species as well as colonization
protective barrier but pellicle provides a
& growth of additional species.
substrate on which bacteria
Microorganisms are generally
progressively accumulate to form Dental
transported passively to the tooth
Plaque. Pellicle provides a medium or
surface by the flow of saliva; few oral
base on which bacteria in the oral cavity
bacterial species are motile (e.g. possess
attach. Pellicle gets easily stained & may
flagella), and these are mainly located
display many colors ranging from white
subgingivally. Plaque may be readily
to dark brown due which the teeth appear
visualized on teeth after 1 to 2 days with no
discolored. oral hygiene measures.

Mechanism of Pellicle formation

The enamel surface has predominance
of negatively charged phosphate
groups that interact directly or
indirectly with positively charged
components of salivary & crevicular
fluid macromolecules. Within few
hours bacteria are found on the pellicle.
These initial bacteria colonizing the
pellicle-coated tooth surface are
predominantly gram-positive
microorganisms such as Actinomyces
viscosus & Streptococcus sanguis.
These initial colonizers adhere to the
pellicle through specific molecules
termed adhesins, on the bacterial
surface that interact with receptors in
dental pellicle. The major constituents
of pellicle are salivary glycoproteins,
material and methods
This was laboratory experiments
research conducted at the
Phytochemistry Laboratory of one of
the Arab universities, siwak was
collected from the S. persica tree in
Makkah, Saudi Arabia. After a 5-day
journey from Makkah to the Karolinska
Institute, it was vacuum packed and stored
at -80C. Standardized siwak pieces were
prepared using sticks 5 mm wide. The
sticks were cut into pieces weighing 0.015,
0.03, 0.07, or 0.14 g. The larger pieces (0.07
and 0.14 g) were tested against all bacteria,
whereas the smaller pieces (0.015 and
0.03 g) were tested against P. gingivalis
only. The outer layer of the siwak (the cork,
Fig. 1) was removed just before weighing
and testing.
 S. mutans and L. acidophilus were grown
for 2 days under anaerobic conditions on
Colombia base agar supplemented with
0.01% tryptophan and citrated horse
blood (5%).

A. actinomycetemcomitans was grown

Figure.1
oss section of the siwak showing the different layers of the root
k (a), cortex (b), phloem (c), xylem (d), and pith (e)
Bacterial Strains and Cultivation
The following bacterial strains were
selected for antimicrobial testing of the
siwak:
S. mutans , L. acidophilus , A.
actinomycetemcomitans ,P. gingivalis
,and H. influenza
for 2 days on the same medium but was
incubated in 95% air and 5% carbon
dioxide. P. gingivalis was grown
anaerobically for 4 days on Colombia base
agar supplemented with hemin (0.05
mg/ml), vitamin K (0.01 mg/ml), and
citrated horse blood (5%). H. influenza
was grown for 1 day in 95% air and 5%
carbon dioxide on hematin-agar
supplemented with hemoglobin (1%) and
isovitalex
Table.1
Growth Inhibition (cm) of Different
Bacteria With Various siwak Weights

Bacterial Strain -0.14g Miswak in P Value of

Agar (median Embedded and Suspended Miswak
[range]) Suspended
0.14g (median
Miswak
Suspended Miswak P Value 0.07g (median
[range] [range])

A.actinomycetemcomitans 10.9 (10.0 to 14.0) 0.008 13.0 (12.0 to 14.0) 0.021 8.0 (7.8 to 8.2)
P. gingivalis 14.0 (11.0 to 14.0) 0.370 14.0 (14.0 to 14.0) 0.008 5.0(5.0 to 5.0)
S. mutans 3.2 (2.6 to 4.6) 0.001* 0.46 (0.2 to 1.00) *0.021 No inhibition
L. acidophilus 1.4 (1.2 to 1.6) 0.007 No inhibition — Not tested
H. influenzae 9.3 (5.1 to 11.5) 0.001 11.5 )12.0 to 13.0( 0.008 8.8 (7.2 to 9.0)

Preparation of Bacterial The suspension of S. mutans and L.

Suspensions acidophilus was prepared in phosphate
buffered saline. A.
actinomycetemcomitans and H. influenza
were suspended in Haemophilus test
medium broth. P. gingivalis was
suspended in peptone yeast glucose
media, prepared anaerobically. The
turbidity of all suspensions was
standardized for each bacterial strain
using a spectrophotometer. The
suspensions were adjusted to give ;
3 . 10*8 colony forming units/ml. Each
bacterial suspension was swabbed over
the surface of the special agar plate.
Antibacterial Testing
Two types of tests were conducted. In the
first test, a standardized round hole, 5
mm in diameter, was punched in the
middle of each inoculated agar plate, and
a 0.14 g siwak piece was placed in the
hole. This test was repeated 10 times for
each strain. The second test was designed
to determine the presence of volatile, air-
borne antibacterial compounds. A 0.14 g
siwak piece was suspended with thread 3
mm above each inoculated agar plate.
This test was repeated five times for each
bacterial strain. The inoculated plates
were incubated as described above, and
growth inhibition was evaluated. Two
plate sizes, 8 and 14 cm, were used in
both tests, depending on the strength of
the antibacterial effect of the siwak piece
on the bacterial strain. In addition, lower
weights of siwak pieces were tested in
duplicate in the second test.
Results
the siwak had powerful inhibitory effects
on the growth of P. gingivalis,
A. actinomycetemcomitans, and H.
influenza, less effect on S. mutans, and
much less effect on L. acidophilus. The
0.14 g suspended siwak exhibited
significantly greater inhibition on A.
actinomycetemcomitans and H. influenza
than the siwak embedded in agar (P≤0.01
and P≤0.001 respectively) The 0.14 g
suspended siwak was less effective than
the corresponding embedded siwak on S.
mutans (P ≤0.001) and it had no
inhibitory effect on L. acidophilus (Table
1). The 0.07-g suspended siwak pieces
exhibited significantly less inhibition of A.
actinomycetemcomitans and H. influenza
than the 0.14 g suspended piece (P≤0.05)
, and it had no observable effect on S.
mutans. P. gingivalis was most sensitive
to the siwak piece; the least sensitive was
L. acidophilus. The overall differences
among inhibition zones around P.
gingivalis associated with siwak pieces of
different weights were significant (P =
0.011).
Statistical Analysis
The data were not distributed normally,
and non-parametric statistical tests were
used. The Mann-Whitney U test was used
to compare the inhibitory effects of 0.14 g
siwak samples embedded in inoculated
agar plates and samples suspended above
the plates. It was also used to compare
between the inhibitory effects of 0.14 g
and 0.07 g siwak samples suspended
above the inoculated agar plates. The
Kruskal-Wallis test was used to compare
the inhibitory effects of four different
weights of suspended siwak on P.
gingivalis. The data were analyzed using a
statistical program ,P≤ 0.05 was
considered statistically significant.
 14 16

mean inhibition (cm) (g)

14
12
10
8
5 6
4
1.5
0 2
0
1
weight (g)

1‫سلسلة‬ 2‫سلسلة‬ 3‫سلسلة‬ 4‫سلسلة‬

Figure.2

Correlation of P. gingivalis growth inhibition to siwak of various weights suspended

with thread 3 mm above the agar plate.

Figure 2 shows the dose response of
different suspended siwak weights
(0.015, 0.03, 0.07, and 0.14 g) on P.
gingivalis. Examples of inhibition zones
associated with different methods of
DISCUSSION
testing the siwak are presented in Figure
3. This figure also shows testing of
different siwak weights and full growth of
bacteria in the absence of siwak.
associated with siwak suspended 3 mm
above the inoculated agar plates,
suggesting the presence of volatile active
antibacterial compounds.

The siwak pieces clearly demonstrated

much stronger inhibitory effects than the
aqueous siwak extract. For example, with
respect to S. mutans, the siwak pieces
caused unexpectedly large inhibition
zones of 3.4 cm (2.6 to 4.6 cm), whereas
our preliminary tests of aqueous siwak
Figure.3 extract yielded an inhibition zone of only
0.2 cm; this result was in accordance with
siwak-induced growth inhibition, all with 14-cm plates.
earlier A) P. gingivalis
studies20-23 growth
in which 50%insiwak
the
Ourabsence
findings of support
siwak. B)the
P. gingivalis
hypothesis inhibition
that with 0.14 gextract
aqueous siwak resulted
piece embedded in agar.
in an inhibition
siwak zone of 0.2 to 0.3 cm. The weak
C) P.stick pieces,
gingivalis withoutwith
inhibition extraction,
0.14-g suspended siwak. D) P. gingivalis inhibition with
have a strong antibacterial antibacterial effect of aqueous siwak
0.07-g suspended siwak E)effect against
A. actinomycetemcomitans inhibition with 0.14-g suspended
most of the bacterial species tested. Equal extract suggested that the active
siwak. F) H. influenza with suspended siwak 0.07g.
or greater antibacterial effects were compounds were not extracted or were
deactivated during preparation of the
crude aqueous extract. Such an extract
probably does not reflect the real
antibacterial activity of siwak.
The antibacterial effect of siwak pieces on
A. actinomycetemcomitans, P. gingivalis,
H. influenza, and L. acidophilus cannot be
compared to that of the crude extract
because, to the best of our knowledge,
there are no published studies on the
effect of siwak extract on these bacterial
strains. It is also difficult to compare the
antibacterial effect of siwak pieces to that
of known antibacterial substances. The
reason of difficulty is that the exact
content and amount of siwak pieces is not
known. Thus, the need to evaluate its
effects using
the standard methods of evaluating
antibacterial substances requires us to
find another method of extraction to get
an active siwak extract. Pharmacologic
studies28-30 showed that it is possible to
obtain essential volatile oil from the
roots, stems, and leaves of
S. persica with steam distillation.
However, no studies were carried out to
test the antibacterial activity of the root
oil on oral bacteria. Gas chromatography–
mass spectrometry analyses of the root
oil revealed that it consists mainly of
benzylisothiocyanate (BIT) (70%),
limonene (9.4%), a-pinene (8.7%), and
flavonoids (2.55%).30,31 Some of these
compounds are known to have
antibacterial activity. Studies31-35 on
BIT and flavonoids separately proved that
they have antibacterial, antifungal, and
antiviral activities.
The siwak exhibited stronger
antibacterial activity against the Gram-
negative bacteria tested in this study than
the Gram-positive bacteria evaluated, as
evidenced by the pronounced differences
in inhibition zones associated with the
Gram-negative species A.
actinomycetemcomitans, P. gingivalis, H.
influenza, and the Gram-positive species
S. mutans and L. acidophilus. A study 35
of the effects of BIT and flavonoids on
Gram-negative and -positive bacteria
showed contradictory results. This may
be due to different assays used to test the
antibacterial effect and to variations
within each assay. Well-standardized
studies are needed to identify which
components of the oil exert an
antibacterial effect against Gram-
negative and -positive species.
Comparison of the effect of suspended
and embedded siwak pieces revealed that
the suspended siwak pieces had similar
or stronger effects on Gram negative
bacteria, whereas the opposite was true
for Gram-positive bacteria: the effect of
the suspended siwak was substantially
reduced. Most probably, the compounds
that affect Gram-negative bacteria are
more volatile than those affecting Gram-
positive bacteria. Studying the properties
of different siwak compounds and their
reactions to surrounding environments is
important to determine the best method
available for testing their antibacterial
activity. The effect of suspended 0.14-g
siwak on P. gingivalis was so strong that
all 14-cm agar plates had complete
growth inhibition. However, our
laboratory facilities did not allow
anaerobic incubation of larger agar
plates. To verify that the strong inhibitory
effect of 0.14-g pieces was a real effect of
the siwak and not an artifact, a dose-
response experiment was conducted.
This experiment showed alinearcor
relation between siwak weight and
inhibitory effect (R2 = 0.99).
H. influenza is a well-characterized upper
respiratory tract commensal of humans
and associated animals. It is a major
CONCLUSIONS
etiologic agent of upper respiratory tract
infections and acute exacerbations of
chronic bronchitis.(36) Initially, H.
influenza served as a control for the
incubation environment of A.
actinomycetemcomitans. However, when
the siwak showed strong antibacterial
effect against the former, it was necessary
to include these results in the study.

S. persica roots contain compounds with strong antibacterial activity against the Gram-
negative bacteria tested in this study and some effect against the Gram positive bacteria.
The oral hygiene benefits of chewing sticks from S. persica may be attributable to the
mechanical removal of plaque as well as the potential inhibitory effects against bacteria
implicated in oral diseases, such as caries and periodontitis. Further investigation is
warranted to determine whether mouth rinses and other oral preparations with
antibacterial effects might be derived from S. persica.
REFERENCES
1. Antibacterial activity of Miswak (Salvadora persica L.) extracts on oral hygiene ;
Article in Journal of Taibah University for Science · December 2015

2. Anders Gustafsson, - Sofrata , Rolf Claesson . Strong Antibacterial Effect of Miswak Against Oral
Microorganisms Associated With Periodontitis and Caries , Article in The Journal of Periodontology
· August 2008

3.Khadija. K. Al-Dulaimy Microbiology. Dental plaque and Dental caries.

4. Arni I. Djais, Vidya Y. Tope. Effectiveness of siwak salvadora persica extract to

aggregatibacter actinomycetemcomitans as one of pathogenic bacteria causing periodontal

diseas , ournal of Dentomaxillofacial Science (J Dentomaxillofac Sci) April 2017

5. Abdul–Ghany Omer, Sozan Muhsin Qarani, Amera Kamal Khalil .In vitro antimicrobial
activity of Miswak extracts against some oral pathogenic isolates , 2010

6. Dr.Punit Vaibhav Patel, Sheela KUMAR Gujjar. Clinical effect of Miswak as an adjunct to
tooth brushing on gingivitis, Article in Journal of Indian Society of Periodontology · March
2012.