From the Division of Periodontology, Department of Dental

Medicine
Karolinska Institutet, Stockholm, Sweden

SALVADORA PERSICA
(MISWAK)
An effective way of killing
oral pathogens

Abier Hamed Sofrata

Stockholm 2010
Opponent
Associate Professor Per Ramberg, Department of Periodontology, Institute of
Odontology at Sahlgrenska Academy, University of Göteborg, Göteborg, Sweden.

Examining committee
Professor Gunnar Dahlén, Department of Oral Microbiology, Institute of Odontology at
Sahlgrenska Academy, University of Göteborg, Göteborg, Sweden.

Associate Professor Anette Oliveby, Division of Cariology and Endodontology,

Department of Dental Medicine, Karolinska Institutet, Stockholm, Sweden.

Associate Professor Helene Thorstensson, Institute for Postgraduate Dental Education,

Jönköping University, Jönköping, Sweden.

Supervisors
Professor Anders Gustafsson, Division of Periodontology, Department of Dental
Medicine, Karolinska Institutet, Stockholm, Sweden.

Professor Peter Lingström, Department of Cariology, Institute of Odontology at

Sahlgrenska Academy, University of Göteborg, Göteborg, Sweden.

Professor Anna-Karin Borg Karlson, Department of Chemistry, School of Chemical

Science and Engineering, Royal Institute of Technology, Stockholm, Sweden.

All previously published papers were reproduced with permission from the publisher.
Published by Karolinska Institutet.

© Abier Sofrata, 2010

ISBN 978-91-7409-915-7

Printed by
Printed by
2010

Gårdsvägen 4, 169 70 Solna

To my father Hamed, and to the memory
of my mother Abla,
who was the greatest inspiration of my life.
ABSTRACT

Mechanical removal of dental plaque is regarded as an effective means of controlling

the progression of dental caries and periodontal disease. The habitual use of chewing
sticks (Miswak) as an oral hygiene method is still wildly spread throughout parts of
Asia, Africa, and the Middle East. Thus, the World Health Organization recommends
and encourages the use of chewing sticks as an effective oral hygiene tool in areas
where it is customary. As Salvadora persica (Arak) is one of the most important
species still used around the world, the main aim of this thesis was to test the
hypothesis that Salvadora persica Miswak offers a unique combination of mechanical
and chemical supragingival plaque control.

This thesis was based on two parts. A clinical part (Papers I & IV) investigated the
effect of rinsing with Miswak extract on plaque pH after acidogenic challenge, and the
effect of fresh and deactivated Miswak sticks on dental plaque and gingival
inflammation in patients with gingivitis. A laboratory part (Papers II & III) tested the
antibacterial activity of Miswak pieces and Miswak essential oil obtained by steam
distillation against some Gram positive/negative bacteria. Through Gas
Chromatography-Mass Spectrometry and Medium Pressure Liquid Chromatography
the main antimicrobial component/s of Salvadora persica Miswak was specified and
characterised.

The main findings were both fresh Miswak and essential oil Miswak extract had a
strong antibacterial activity against Gram negative bacteria including some oral
pathogens such as Porphyromonas gingivalis and Aggregatibacter
actinomycetemcomitans. Benzyl isothiocyanate (BITC) was the main antimicrobial
component of Miswak. Bacterial cell membrane exhibited protrusions upon treatment
with Miswak oil and commercially available BITC. Rinsing with Miswak extract,
compared with water rinsing, resulted in protracted elevation of plaque pH (>6.0). Both
fresh and deactivated Miswak reduced dental plaque and gingival inflammation. The
decrease was statistically significant with active Miswak but was not with deactivated
Miswak.

The positive clinical effects of Miswak on plaque pH, plaque and gingival indices, as
well as the immediate strong antibacterial activity demonstrated in the laboratory
experiments suggested that Miswak could be beneficial for oral health. Further
laboratory and clinical investigations of antiviral and antifungal activities together with
the effect on periodontal inflammation need to be performed. The cytotoxic activities of
fresh Miswak and Miswak oil need to be evaluated before the development of oral
applications becomes a future reality.

Key words: Miswak, Salvadora persica, chewing stick, traditional medicine, gingivitis,
periodontitis, dental plaque, plaque pH, salivary flow, acid production, microelectrodes,
oral hygiene, subgingival microbiota, antibacterial agent, isothiocyanate,
chromatography, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans.
LIST OF PUBLICATIONS
This thesis is based upon the following papers, which will be referred to in the text by
their Roman numerals:

I. Sofrata, A., Lingström, P., Baljoon. M., Gustafsson, A., 2007. The effect of
Miswak extract on plaque pH. Caries Research 41(6): 451-454. (Clinical
Paper)
II. Sofrata, A.H., Claesson, R.L., Lingström, P.K., Gustafsson, A.K., 2008.
Strong antibacterial effect of Miswak against oral microorganisms associated
with periodontitis and caries. Journal of Periodontology 79, 1474-1479.
(Laboratory Paper)
III. Sofrata, A., Santangelo, E., Azeem, M., Borg-Karlson, A.-K., Gustafsson, A.,
Pütsep, K., 2010.Benzyl isothiocyanate the main antibacterial component of
Miswak sticks is highly active against the Gram-negative proteobacteria.
(Laboratory Paper). Submitted
IV. Sofrata, A.H., Brito, F., Al-Otaibi, M., Gustafsson, A., 2010.Short term
clinical effect of active and inactive Salvadora persica Miswak on dental
plaque and gingivitis. (Clinical Paper). Manuscript
CONTENTS
INTRODUCTION ........................................................................................................................................1

HISTORY OF TOOTH CLEANING AIDS ...........................................................................................................1

MISWAK: THE NATURAL TOOTHBRUSH ......................................................................................................2
SALIVA BUFFERING CAPACITY AND ANTIMICROBIAL COMPOSITION ..........................................................5
PLAQUE pH MEASUREMENTS .....................................................................................................................6
MECHANICAL AND CHEMICAL PLAQUE CONTROL .......................................................................................7

AIMS.............................................................................................................................................................10

MISWAK USED IN ALL FOUR STUDIES ...........................................................................................11

CLINICAL PART (PAPERS I & IV) ......................................................................................................11

MATERIALS AND METHODS ..............................................................................................................11

PAPER I .....................................................................................................................................................11
PAPER IV ...................................................................................................................................................14

RESULTS.....................................................................................................................................................17

PAPER I ......................................................................................................................................................17
PAPER IV ...................................................................................................................................................21

DISCUSSION ..............................................................................................................................................23

LABORATORY PART (PAPERS II & III)............................................................................................26

MATERIALS AND METHODS ..............................................................................................................26

PAPER II.....................................................................................................................................................26
PAPER III ...................................................................................................................................................28

RESULTS.....................................................................................................................................................38

PAPER II.....................................................................................................................................................38
PAPER III...................................................................................................................................................40

DISCUSSION ..............................................................................................................................................51

MAIN FINDINGS.......................................................................................................................................55

CONCLUSIONS .........................................................................................................................................56

FUTURE PERSPECTIVES ......................................................................................................................56

ACKNOWLEDGEMENTS.......................................................................................................................58

REFERENCES............................................................................................................................................60
LIST OF ABBREVIATIONS
AP Approximal plaque
BHI Brain heart infusion
BITC Benzyl isothiocyanate
CFU Colony forming units
DMC Dichloromethane
DMFT Decayed, Missing, and Filled Teeth
DMSO Dimethyl sulfoxide
Dsb Disulfide bond (protein family)
EtOAc Ethyl acetate
EtOH Ethanol
GC-MS Gas Chromatograph-Mass Spectrometry
GI Gingival index
H2O2 Hydrogen peroxide
HCl Hydrochloric acid
He Helium, Carrier gas
hMPO Human myloperoxidase
hSPO Human salivary peroxidase
Mg2So4 Magnesium sulphate
MPLC Medium Pressure Liquid Chromatography
NA Not applicable
NIST National Institute of Standards and Technology library
PBS Phosphate Buffer Solution
pH Potential for hydrogen ion concentration
PI Plaque index
PYG Peptone yeast glucose medium
Rf Retardation factor
S. persica Salvadora persica
SCN– Thiocyanate
SPME Solid phase microextraction
TLC Thin layer chromatography
INTRODUCTION

HISTORY OF TOOTH CLEANING AIDS

Man has long been interested in his appearance and maintaining a clean, pleasant
appearing mouth and a nice smile. Tooth cleaning aids and toothpicks are traceable
back in history (Hyson, 2003). Chewing sticks were used by the Babylonians as early
as 3500 BC. Ancient Greek and Roman literature discusses toothpicks that were
chewed to help cleaning the teeth and the mouth (Wu et al., 2001). Hippocrates (355
BC) recommended a round woollen ball dipped in honey as a toothbrush. Romans also
used toothpicks from the mastic tree to clean their teeth (Hyson, 2003). Ancient Arabs
were accustomed to Miswak to get their teeth white and shiny (Bos, 1993).
This kind of toothbrush was not just used by the Arabs, others used something similar,
for example, the Japanese called it Koyoji and the Jews used a kind of wooden stick
called Qesam in Hebrew and was mentioned in the Talmud. Even in the 1920s in some
rural areas of the United States, chewing stick made of dogwood was still used (Hyson,
2003). In comparison with the chewing stick, the conventional toothbrush is relatively
modern. According to the 17th century Chinese encyclopaedia, the first toothbrush was
made in China in 1498. Even as late as the 1750s in Europe, the toothbrush appears
relatively uncommon, although the toothpick is mentioned in the literature of the day.
The toothbrush was strictly a "novelty sold in Paris for the cleaning of the teeth"
(Hyson, 2003). Moreover, there is no illustration of an American toothbrush dated
before 1818 (McCauley, 1946).

Today, manual toothbrushes are available at affordable prices for almost everyone,
although many people in Afro-Asian communities cannot afford to purchase
commercial toothbrushes and dentifrices. Miswak not only offers economical cleaning
of the teeth, but has been traditionally used by many cultural groups for centuries, and
religion reinforced these traditions. The influence of Islam on the spread and use of
chewing sticks in different parts of the world is important (Khoory, 1983). Islam
reinforced the use of Miswak as an aspect of dental care. Muslims follow the example
of their prophet, and according to him, the Miswak should be used five times a day
before each prayer (Bos, 1993). A national health survey in Pakistan found more than
half of the rural population use chewing sticks as an oral hygiene tool (Asadi and
Asadi, 1997). Several studies investigating oral hygiene habits in Saudi Arabia
highlight the use of Miswak is popular as an oral hygiene aid in different population
categories (Almas et al., 2003a; 2003b; 2003c; 2003d). However, among Saudi
Arabians, there are large differences in current oral hygiene habits, which are mainly
related to age and socio-economic level, and to a lesser extent gender (Al-Otaibi et al.,
2003). In Africa, the use of chewing sticks is still wide spread, and chewing sticks are
widely used in Sudan, Nigeria, and Namibia: many studies have been based on
chewing sticks widely used in these countries (Wolinsky and Sote, 1983; Cai et al.,
2000; Darout et al., 2000a).

1
MISWAK: THE NATURAL TOOTHBRUSH

Miswak: Definition and use

The name Miswak, also called miswaak, Miswaki, siwak, siwaki depending on the
Arabic dialect and the country, is known in English as the natural toothbrush (Elvin-
Lewis, 1980; Hattab, 1997; Al-Sadhan and Almas, 1999). Throughout the world, 182
species of plants have been used as chewing sticks, with 158 known to Africa alone. In
Ghana and Nigeria, Teclea vardoordniana, Garcinia, and Acacia species are preferred.
Azadirachta indica (Neem tree) is the most popular species used in India, Pakistan, and
Nepal. In the Americas, Cornus florida (Dogwood) was used for many dental purposes.
The most important species is Salvadora persica (S. persica), also known as Arak, as it
is the main plant used as chewing sticks from East Africa through to the Asian
subcontinent, including Saudi Arabia (Elvin-Lewis, 1980; 1982; Almas and Al-Lafi,
1995).

S. persica is a small tree or a shrub with a crooked trunk; usually one foot in diameter
[Figure 1], the roots are spongy and easy to crush between the teeth. Pieces of the root
usually swell and become soft when soaked in water (Eid et al., 1990b). For the root to
be prepared for cleaning teeth, the stick is typically cut into 15 cm lengths. The width
of the stick is usually from 0.7 to 1 cm wide. The stick is washed with water to free it
from sand and before use, the bark is removed from the functioning end (about 1 cm
long) of the stick, beaten hard or chewed until it becomes frayed [Figure 2]. The
techniques employed to mechanically remove plaque are similar to that of the
toothbrush. The cleaning movement should always be directed away from the gingival
margin of the teeth on both buccal and lingual surfaces. Circular movements can be use
to massage the gum, but care should be taken not to damage the soft tissue of the
mouth. Fresh soft Miswak is preferred: if the Miswak is dry, the end of the Miswak
should be soaked in fresh water for 24 hours before use (Almas and Al-Lafi, 1995;
Almas et al., 1997). After use another brush is made from the rest of the stick. It is
recommended to renew the head of the Miswak and use a fresh head as often as
possible. The optimal way is to use the crushed part until it loses its taste and odour. At
that point, the overused end has to be cut off because the presence of odorous
components is considered a good indicator for tooth brushing efficiency (Bader et al.,
2002). The same head is not recommended to be used for more than 24 hours because
of cytotoxic activity (Mohammad and Turner, 1983). Usually, the teeth are brushed for
5 to 10 minutes, but some users chew on the stick for several hours while involved in
other activities: this habit lead to the name “chewing stick” (Hattab, 1997). However,
the term chewing stick is considered misleading as the stick is chewed only briefly to
fray its fibres before its common use as a toothbrush (Gazi et al., 1990).

2
Figure 1: Salvadora persica (Arak) shrub growing naturally in the desert.

B C

Figure 2: Miswak chewing sticks. A) Miswak before preparation for use. B) Frontal view of
the Miswak bristles used for bushing teeth. C) Lateral view of the Miswak showing the
prepared head for brushing teeth.

Different Miswak extracts and reported antibacterial activity

Miswak from S. persica chewing sticks has been tested for its antibacterial activity by
different antimicrobial testing methods. The forms where Miswak was extracted are
also different, including water, ethanol, methanol, ethyl acetate, and acetic acid extracts
(AbdElRahman et al., 2002; Noumi et al., 2010). The usual method of crude Miswak
extract preparation is by sun drying the sticks for few days. The sticks are then cut into
small pieces and ground to powder with a mill or a household grinding machine. The
powder is then mixed with sterile deionised water (or one of the previous mentioned
solvents). The extract is allowed to soak for 48 hours. Then the mixture is centrifuged
and filtered. Different dilutions of this extract are then prepared and tested against

3
different microorganisms (Al-Lafi and Ababneh, 1995; Almas et al., 1997; Almas,
1999; Almas, 2001).

In vitro studies testing this crude Miswak extract conclude Miswak has considerable
antibacterial effect, which increases with extract concentrations (Al-Lafi and Ababneh,
1995; Almas et al., 1997). Water Miswak extract has an inhibition zone of 20 mm on
Streptococcus mutans and 24 mm on Staphylococcus aureus (Al-Lafi and Ababneh,
1995). In other studies with water extract, inhibition zones on Streptococcus faecalis of
1 mm (5% Miswak extract), 4 mm (10% Miswak extract), and 7 mm (50% Miswak
extract) are reported: the 50% extract had 3 mm inhibition zone on S. mutans (Almas,
1999; Almas and Al-Bagieh, 1999; Almas, 2001). Aqueous Miswak extracts of 15%
and 20% concentrations have a fungistatic effect on Candida albicans for up to 48
hours (Al-Bagieh et al., 1994). Different alcohol extracts of S. persica have potent
antifungal activity on different Candidal species. In a study on different extraction
solutions, ethanol extract of Salvadora persica root is the most potent and S. mutans is
the most susceptible strain (AbdElRahman et al., 2002). However, the results from
these studies cannot be compared as the Miswak sources and the concentrations
predations are different.

A volatile oil has been obtained form Jordanian Salvadora persica leaves, stem, and
root. Gas Chromatography-Mass Spectrometry (GC-MS) analyses of the oil revealed
different main components in different parts of the plant. The leaves have Benzyl nitrile
(54%) and the stem has Cineole (46%) as the main components with reported
antibacterial activity (Alali and Al-Lafi, 2003; Alali et al., 2004). The roots contain
Glucotropaeolin, where Benzyl isothiocyanate and benzyl nitrile are separated by the
enzymatic hydrolysis of the glucosinolate (Ezmirly and Seif-Elnasr, 1981; Bader et al.,
2002). Synthetic Benzyl isothiocyanate has virucidal activity against Herpes simplex
virus, inhibits the growth and acid production of Streptococcus mutans, and is
fungistatic to Candida albicans (Al-Bagieh and Weinberg, 1988; Al-Bagieh, 1992; Al-
Bagieh, 1998). However, the chemical compositions of S. persica roots and the exact
amounts of each component are contradictory (Ezmirly and Seif-Elnasr, 1981; Abdel-
Wahab et al., 1990; Bader et al., 2002).

The clinical effects of Miswak on oral health

When researchers realised large numbers of communities depend on chewing sticks as
the primary oral hygiene method, the urge to identify its effect on dental health arose.
Africa was the first area of focus. Elvin-Lewis et al (1980) evaluated the dental health
of 887 habitual Miswak users in southern Ghana for habits and type of chewing stick
preference. He summaries the findings that the presence of antibacterial and
anticarioginic substances in many of the sticks suggests the low caries rates among
Miswak users may not be related just to efficient plaque removal. A pilot cross-
sectional study in West Africa evaluating the chewing stick compared to the toothbrush
in 163 participants indicated plaque and bleeding scores were higher in chewing stick
users and concluded chewing stick appears to be less efficient than the toothbrush in
plaque removal, and the antimicrobial agents contained in the sticks offer no
advantages over tooth brushing with toothpaste (Norton and Addy, 1989). Later on,
several studies were published about oral health effects of Miswak in different parts of

4
the world. In Saudi Arabia, studies reported Miswak use reduces bleeding and plaque
scores, and Miswak users have similar pocket depths to toothbrush users. However,
Miswak users had significantly higher gingival recession than toothbrush users. The
results of these studies indicate the use of Miswak influences plaque accumulation and
periodontal health. The findings also reveal that the level of need for periodontal care in
the study samples was lower than for toothbrush users in the same communities (Eid et
al., 1990a; Eid et al., 1990b; Al-Khateeb et al., 1991; Eid et al., 1991; Guile, 1992). In a
sample of Sudanese population, the periodontal status of Miswak users is better than
that of toothbrush users, suggesting the efficiency of Miswak use for oral hygiene in
groups familiar with its use, and that Miswak is comparable or slightly better than the
toothbrush (Darout et al., 2000a).

Clinical studies in children have evaluated the efficiency of traditional Miswak as an

oral hygiene tool among various populations and find Miswak as effective as the
toothbrush in removing oral deposits. Miswak reduces plaque and gingival scores to the
same extent as the toothbrush; however, the use of toothpaste with the Miswak does not
increase Miswak effectiveness (Olsson, 1978; Danielsen et al., 1989; Helderman et al.,
1992). Other clinical studies in adults determine Miswak is more effective than the
toothbrush for reducing plaque and gingivitis, and appears more effective in removing
plaque from embrasures, thus, enhancing interproximal health (Gazi et al., 1990; Al-
Otaibi et al., 2003). Miswak has an effect on salivary and subgingival microbiota
levels. Darout et al. (2002; 2003) found that Miswak users have higher levels of some
oral bacterial strains including A. actinomycetemcomitans and lower levels of other oral
bacterial strains than the toothbrush users. However, Al-Otaibi et al. (2004) found
Miswak users had lower A. actinomycetemcomitans levels than toothbrush users. These
findings suggest Miswak may have a selective inhibitory effect on the level of certain
bacterial strains in saliva and plaque. Miswak has an immediate effect on the
composition of mixed saliva and there is a significant increase in calcium and chloride
concentrations when a piece of Miswak is chewed rather than rubber while pH and
phosphate values are significantly lowered (Gazi et al., 1992). There is also an
immediate antibacterial effect of Miswak on saliva with immediate significant
reduction of S. mutans and non significant reduction in Lactobacilli when comparing
Miswak with toothbrush (Almas and Al-Zeid, 2004).

SALIVA BUFFERING CAPACITY AND ANTIMICROBIAL COMPOSITION

Saliva is an essential component required for maintenance of the ecologic balance in

the oral cavity. Saliva does not only lubricate oral tissues, making swallowing and
speaking possible, but also protects oral tissues, teeth, and mucosal surfaces in many
ways. These largely protective functions are best observed in xerostomic patients,
when severe reduction of salivary output occurs (Edgar and O'Mullane, 1996). Saliva
is important in maintaining a neutral pH level in plaque and in the oral cavity while
constantly bathing the teeth and the oral mucosa. Stimulated saliva is a powerful buffer
that plays a critical role in limiting pH changes after acid production by plaque
bacteria. Increased salivary flow results in increased pH and buffering capacity
because of the increased rate of clearance of oral sugar and plaque acid due to higher
salivary film velocity, and the increase in bicarbonate, which is the most important

5
buffering system in the saliva at high flow rates. However, in unstimulated saliva, the
level of bicarbonate ions is too low to be an effective buffer (Dawes and Kubieniec,
2004). The phosphate system has an important role at low salivary rates, where its
concentrations are very high. However, phosphate concentrations become very low
with salivary stimulation (Edgar and O'Mullane, 1996). Other factors such as urea
content of saliva are important as many plaque organisms posses urease activity,
converting urea to ammonia; thus, raising plaque pH (Edgar and O'Mullane, 1996;
Dawes and Kubieniec, 2004).

The antimicrobial functions of saliva are probably one of the most important functions
of saliva. Human mixed whole saliva contains two principal defensive peroxidase
systems in the oral cavity, salivary peroxidase (hSPO) secreted from the acini of human
parotid and submandibular glands, and myloperoxidase (hMPO) from
polymorphonuclear leukocytes coming into saliva from the gingival crevicular fluid.
The major function of hSPO and hMPO is to catalyse the oxidation of thiocyanate
(SCN–) in the presence of hydrogen peroxide (H2O2), resulting in end products with
wide antimicrobial potential (Ihalin et al., 2006; Ashby, 2008). Human salivary
peroxidase systems belong to the group of innate defence factors. The antibacterial
spectrum of salivary peroxidase systems covers both Gram positive and negative oral
and non-oral bacteria (Tenovuo, 1998). In addition to being an antibacterial, the
peroxidase systems have antiviral as well as antifungal activities (Lenander-Lumikari,
1992; Mikola et al., 1995). Salivary peroxidase systems have various antibacterial
mechanisms, some which are bacteriostatic inhibiting bacterial growth, and some of
which are more bacteriocidal, killing the microorganism. Gram negative bacteria are
more susceptible than Gram positive (Marshall and Reiter, 1980; Ihalin et al., 2001),
and anaerobically-grown bacteria are more susceptible than aerobically-grown bacteria
(Carlsson et al., 1983). As human saliva contains a variety of innate defence
mechanisms, which act in the concerted manner and may exhibit synergistic effects
(Lenander-Lumikari, 1992), the role of a single defence factor is difficult to define
(Ihalin et al., 2006).

PLAQUE pH MEASUREMENTS

The oral environment is in continues state of flux, and changes in a large number of
factors can be envisioned to alter the response of plaque to fermentable carbohydrates
There are many host factors influencing plaque response to fermentable carbohydrates
such as buffering capacity of the saliva, plaque, and ions released from enamel; calcium
and phosphate concentration of saliva; flow rate and viscosity of saliva; presence and
age of the plaque at caries prone sites in the dentition; components of the plaque matrix
affecting diffusion; anatomy of the dentition; microstructure of enamel; fluoride content
of enamel and plaque; pattern of mastication, sucking, rinsing, and swallowing; and the
frequency of food ingestion(Schachtele and Jensen, 1982).

There are three different methods used to assess plaque pH: the sampling, the
microtouch, and the telemetric. The advantages and limitations of each method are
discussed in different studies.

6
The Sampling method
The Sampling method involves repeated removal of small samples of plaque from a
number of teeth at intervals after food ingestion, dispersion of the sample, and in vitro
measurement of pH. This method does not require sophisticated equipment, and is
efficiently used on a large number of subjects. However, the main disadvantage is that
plaque sampling must be critically timed unless bacterial metabolism is blocked after
plaque removal.

The Telemetric method

This is the most sensitive method for measuring changes in plaque pH. It involves the
placement of glass microelectrodes or ion-sensitive field effect transistors within the
dentition. Plaque is allowed to accumulate on the microelectrodes, and the pH changes
are subsequently transmitted by radio or wire. The main advantages of this method are
continuous monitoring of the pH of undisturbed plaque and the ability to study older
plaque (i.e. weeks to months). However, technically, it is more demanding than other
methods as it requires sophisticated equipment, patient selection is limited by the need
for appropriate space in the dentition, and the microbial content of plaque on sensing
surface might not be normal.

The Microtouch method

This method uses glass or antimony microelectrodes, which are placed onto plaque in
situ where direct readings can be obtained. The main disadvantage is that some
electrodes are fragile and lack stability. However, this method had the requirements
needed for our study in that it does not require any sophisticated equipment and could
be efficiently used on a large number of subjects with numerous individual accessible
sites that can be monitored with few seconds between them (Jensen et al., 1982;
Schachtele and Jensen, 1982; Yamada et al., 1982; Harper et al., 1985; Harper et al.,
1986).

MECHANICAL AND CHEMICAL PLAQUE CONTROL

Plaque formation starts almost immediately after a tooth surface has been cleaned.
Saliva is seldom in direct contact with the tooth surface. A thin layer of 10-100μm
thick acellular layer of absorbed salivary proteins and other macromolecules, called the
acquired pellicle, separates saliva from the tooth surface. This thin layer forms the base
for subsequent adhesion of microorganisms, and is then termed dental plaque (Gibbons,
1984; Fejerskov and Kidd, 2003). For descriptive purposes plaque development is
divided into three phases: phase ȱ, phase ȱȱ, and phase ȱȱȱ (Fine, 1995; Lindhe et al.,
2003). Today, there is strong evidence that both dental caries and periodontal diseases
are associated with oral microorganisms, in other words, dental plaque (Löe et al.,
1965; Lindhe and Axelsson, 1973). The concept of anti-plaque agents can be used in
one of the different phases of plaque development: to interfere with the adhesion of oral
bacteria to surfaces and prevent biofilm formation; to interfere with co-aggregation
mechanisms or to affect bacterial vitality, which thereby prevents future growth of
colonies; or, to remove or disrupt existing biofilms (Baehni and Takeuchi, 2003).

7
In periodontal disease treatments, some cases progress even after mechanical removal
of plaque, thus, conventional periodontal treatments are insufficient for preventing
progression of the disease (Hirschfeld and Wasserman, 1978; Isidor and Karring, 1986;
Fine, 1995). In such cases, additional chemical plaque control could be of importance
(Löue et al., 1976; Gordon et al., 1985; Grossman et al., 1986; DePaola et al., 1989;
Svatun et al., 1990). Three of the most commonly used antimicrobial agents are
chlorhexidine, essential oil (EO), and triclosan (Fine, 1995). At low concentrations,
chlorhexidine acts as a bacteriostatic and damages the bacterial cell membrane: at high
concentrations chlorhexidine causes precipitation and coagulation of the bacterial
cytoplasm leading to bacterial death (Grossman et al., 1986; Jones, 1997; Baehni and
Takeuchi, 2003). EO mouthwashes are also important antiplaque agents (Ouhayoun,
2003). Essential oil acts on microorganisms by disrupting their cell wall and inhibiting
enzymatic activity (Fine et al., 1985; Gordon et al., 1985; DePaola et al., 1989). The
advantage of EO mouthwashes over chlorhexidine is the ability to penetrate plaque
biofilm (Ouhayoun, 2003). Triclosan affects microorganisms in a similar manner to EO
(Fine, 1995).

The majority of the bacteria in the biofilm on teeth may be considered commensal
bacteria. Commensalism is the situation when one population or individual (the
bacteria) benefit from the relationship while the other (the host) neither benefits nor is
harmed. In periodontal diseases, the biofilm life-style is advantageous to some bacteria;
and if they are not removed by hygiene measures, they may expand and cause disease.
These bacteria have thus become opportunistic pathogens. In the oral cavity, bacteria
such as Porphyromonas gingivalis and Streptococcus mutans are opportunistic
pathogens. This is contrary to pathogenic bacteria that never constitute part of the
commensal community of a host and always cause disease to the particular host. One
such example of a pathogenic bacterium is Salmonella entiritidis, which may invade
the human intestine. The border between commensals and opportunists (also called
conditional pathogens) is sometimes vague, essentially when a group of bacteria
expand in a bacterial community out of the host control, there is always a risk the
bacteria may cause disease to the host (Samaranayake, 2006).

In this thesis the intestinal pathogen Salmonella Typhimurium, the opportunistic

pathogens Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes,
Haemophilus influenzae as well as the commensals Escherichia coli (two different
strains), Enterococcus faecalis, Enterococcus faecium, Lactobacillus fermentum, and
the oral opportunistic pathogens Aggregatibacter actinomycetemcomitans,
Porphyromonas gingivalis, Streptococcus mutans and Lactobacillus acidophilus were
used. Dental caries is a multifactorial disease in which, among others, mutans
streptococci (S. mutans and S. sobrinus) and lactobacilli, separately or together, play an
important role (Featherstone, 2000; Selwitz et al., 2007). Increased levels of mutans
streptococci are strongly associated with caries initiation, and lactobacilli are strongly
associated with deep carious lesions requiring restoration (Beighton et al., 1996; Ajdic
et al., 2002; Fejerskov and Kidd, 2003). The World Workshop in Periodontics (report.,
1996) reported there is sufficient evidence to incriminate P. gingivalis, Tannerella
forsythia (previously T. forsythensis), and A. actinomycetemcomitans (previously
Actinobacillus actinomycetemcomitans) as causative factors for periodontitis. Studies
show that strains of A. actinomycetemcomitans and P. gingivalis are capable of

8
invading epithelial cells derived from human epithelial periodontal pockets or gingival
sulci (Fives-Taylor et al., 1995; Dierickx et al., 2002; Andrian et al., 2006). A.
actinomycetemcomitans also produces leukotoxin that lyses polymorphonuclear
leukocytes and monocytes, which enables it to evade the innate defence line of the
periodontal pocket (Johansson et al., 2000). P. gingivalis is characterised by production
of an unusually extensive array of proteases, collectively designated the gingipains.
These gingipains play major roles in P. gingivalis colonisation, host tissue invasion,
destruction of collagen and fibrinogen, and hydrolysis of haemoglobin (Brien-Simpson
et al., 2003).

This research was directed towards the field of natural antibacterial substances. Plants
have innate protective mechanisms to protect against microbial attacks. Many
toothbrush plants have been used in traditional medicine to treat diseases other than oral
diseases. Chewing sticks or Miswak as a traditional cleaning toothbrush might offer
simultaneous mechanical and chemical plaque control.

9
AIMS
Overall aim
The main aim of this thesis was to test the hypothesis that Miswak offers a unique
combination of mechanical and chemical supragingival plaque control.

Specific aims

Clinical part
x To document the changes in plaque pH when an acidic challenge was followed
by rinsing with water Miswak extract.
x To evaluate the effect of Miswak rinse on parotid gland secretion rate.
x To evaluate the effect of active and inactive Miswak on dental plaque,
subgingival microbiota, and gingival inflammation in patients with gingivitis.

Laboratory part
x To test in vitro the antibacterial effect of Salvadora persica Miswak pieces,
without extraction, on bacteria implicated in the etiology of periodontitis and
caries.
x To find an extract of Miswak that could show the strong antibacterial activity
achieved by Miswak pieces.
x To test these extracts for an antimicrobial effect similar to the stick on a range
of Gram negative and Gram positive bacteria.
x To identify, characterise, and isolate the active compound or compounds found
in Miswak.

10
 MISWAK USED IN ALL FOUR STUDIES
The Miswak used in all four studies was roots of the Salvadora persica shrub. Miswak
was bought fresh from the local market in Makkah city, Saudi Arabia. Identification of
the plant as Salvadora persica (Arak) was performed by a professional Arak seller and
by Meshari Al-Otaibi (one of the authors of Paper IV). The Miswak used in Papers I, II,
and III was sent on a 5-day journey from Makkah to the Karolinska Institute, Sweden,
where it was vacuum packed and stored at –80°C until it was used. The Miswak in
Paper IV was used fresh with no storage, as the study was performed in Makkah.

CLINICAL PART (PAPERS I & IV)

The two clinical papers included participants who used Miswak in two different ways:
a rinse of a Miswak water extract prepared immediately before use (Paper I), and fresh
or deactivated Miswak used as a toothbrush (Paper IV). The clinical effects indicated
Miswak is viable for further investigations and presents opportunity for new research
fields.

MATERIALS AND METHODS

PAPER I

Miswak water extract preparation

Miswak was used as a water extract mouth rinse. Fresh Miswak was cut into small
pieces and ground with a grinding machine into a wet powder. The fresh powder was
mixed with distilled water, 5g of Miswak to 20 ml of distilled water, to give a 20%
(w/w) concentration. The mixture was allowed to soak for one hour at 4°C and then
centrifuged at 2500 rpm (1400 gravity) for 10 min. The remaining pellet was
centrifuged for another 5 min to remove the remaining liquid. The supernatants were
passed through filter paper (class1 No.00M) and the extract was used immediately after
filtration.

Laboratory experiment testing the buffering capacity of Miswak water extract

Thirty ml of 20% (w/w) freshly prepared Miswak extract was titrated with 0.02%
hydrochloric acid (HCl), pH 2.30. The pH of the Miswak extract was measured, and
then every five minutes, 0.5 ml of Hydrochloric acid (HCl) was added to the Miswak
extract while the mixture was continuously stirred, and then pH was measured.

Participants
Ten volunteers (4 men and 6 women) aged 30 to 45 years (mean 35.2 years)
participated in the study. The mean Decayed, Missing, and Filled Teeth (DMFT)
(13.6), mean stimulated whole saliva flow rate (1.66 ml/min), mean saliva buffer
capacity (4.28 pH units) were registered. The buffering capacity was measured

11
according to Ericsson (1959). Participants had good general and good oral health, and
had •20 natural teeth, and did not wear orthodontic appliances or removable partial
dentures. None of the participants were on any medications. All participants gave their
informed consent. This study was approved by the Regional Research Ethics
Committee at Karolinska Institute, Stockholm, Sweden (Registration No. 2005/76-31).

Study design
The participants were instructed to refrain from tooth brushing, from all other oral
hygiene procedures, and from chewing gum for three days before start of the study.
They were instructed not to eat or drink anything but water in the last three hours
before each test session. Each participant attended three sessions; one for testing the
Miswak oral rinse, one for water rinse (placebo), and a third session with no activities
after the sucrose rinse (control) [Figure 4]. The three test sessions were in a randomised
order. Plaque pH was measured by the microtouch method (Lingström et al., 1993a)
[Figure 3]: an iridium touch microelectrode* (0.1mm in diameter) was inserted into the
interproximal plaque, under the contact area in the right and left maxillary premolar
regions. For each test session, plaque pH was measured at baseline (0 min). The subject
then rinsed for 2 min with 15 ml of 5% (w/w) aqueous sucrose rinse. Nine min after the
sucrose rinse, the subjects were given either a) a rinse with 15ml of 20% Miswak
extract, or b) a rinse with 15ml of water, or c) no rinse. The rinses were performed for 2
min. Plaque pH was measured at set time points from the start to the end of the
experiment: 0, 2, 5, 8, 13, 15, 30, 45, 60 min [Figure 4].

Figure 3: Photograph of the microtouch instrument, to the right, an enlarged picture of iridium
touch microelectrode with a diameter of 0.1mm.

*
Beetrode; WPI Instruments, Sarasota, FL, USA.

12
 10 Participants refrained
from tooth brushing
for 3 days

Baseline plaque pH
measurement

5% sucrose rinse
for 2 minutes

At min 9

No further activities Rinsing with 15 ml Rinsing with 15 ml

after sucrose rinse miswak extract water

Plaque pH was measured

at the following time points

0, 2, 5, 8, 11, 13, 15, 30, 45, 60

minutes

Figure 4: The general plan of the study.

13
In a separate session, the effect of Miswak rinse on parotid gland secretion rate was
tested. Unstimulated parotid saliva was collected for 7 min with a modified Carlson-
Crittenden device [Figure 5] (Shannon et al., 1962). Then parotid saliva was collected
during stimulation with Miswak extract rinse for 2 min, and during the 5 min directly
after the rinse (in total for 7 min).

Figure 5: Modified Carlson-Crittenden device.

Statistical analyses
Two-way repeated-measures ANOVA revealed a significant method×time interaction.
Thus, comparisons between methods were performed at each time point. Paired t-tests
were used to compare plaque pH readings between right and left sides, and to compare
stimulated and unstimulated parotid saliva flow rates. The data were analysed using the
STATISTICA (7.0) program*. P < 0.05 was considered statistically significant.

PAPER IV

Miswak
Active Miswak was used fresh form the market. Inactive Miswak was obtained by
boiling the fresh Miswak sticks in water for two hours. Deactivation was confirmed by
in vitro antibacterial testing on H. influenzae (ATCC 49247). H. influenzae was grown
and tested with active and inactive Miswak [Figure 6]. Each subject was provided with
six Miswak sticks 20 cm in length and 7mm in width and they were instructed to keep
the sticks in the refrigerator until use.

*
StatSof, Inc. Tulsa, Oklahoma, USA.

14
Figure 6: Bacterial growth with active and inactive Miswak. A) Total bacterial inhibition with
active Miswak. B) Complete bacterial growth with inactive Miswak.

Participants
Sixty eight (21 males and 47 females) regular dental patients at the Security Forces
Polyclinics, Dental Department, Makkah City, Saudi Arabia, were invited to participate
in the study. To be included, the patients had to be •18 years of age and have at least 24
teeth. Exclusion criteria were systemic disease, long term medication, antibiotics during
the last 6 months, and pregnancy. Participants with gingival pockets >5 mm and/or
orthodontic appliances were also excluded. All participants were familiar with the use
of Miswak and gave their informed consent. The participants were asked to refrain
from Miswak use for two weeks prior to the study start. The study was approved by the
General Medical Affairs Administration at Makkah City and the regional Research
Ethics Committee in Stockholm (Registration No. 2009/1077–31/4).

Study design
This study was a double-blind, randomised clinical trial. The participants were
randomised by assigning an even or odd number by the random binary outcome of the
toss of a dice. Participants with odd numbers were assigned to active Miswak (test
group) and participants with even numbers were assigned to the inactive Miswak
(control group).

One week before the start of the study, the participants received an intraoral
examination, scaling, and professional tooth cleaning. The volunteers were asked to
continue their usual oral hygiene during the following week.

One week later the participants underwent baseline clinical examination and samples of
subgingival microflora were collected. The participants then received either active or
inactive Miswak sticks according to their assigned group. They were instructed to use
the respective Miswak five times a day over the subsequent three weeks, and not to use
a toothbrush, inter dental cleaners, chewing gum or any other Miswak apart from the
one given, during the time of the study. After three weeks, a follow-up examination and
sample collection were performed in the same manner as at baseline. Neither the
participants nor the dentist knew to which group the participant belonged. The dental
assistant controlled randomisation and grouping.

15
Clinical Examination
The clinical parameters measured were: a) modified gingival index (GI) (Löe and
Silness, 1963; Löe, 1967), and b) plaque index (PI) according to Turesky modified
Quigley-Hein plaque index (Quigley and Hein, 1962; Turesky et al., 1970). Both
gingival inflammation and plaque were registered at four sites per tooth (buccal, mesial,
distal, and lingual) for all teeth, except the third molar. Plaque was stained with
erythrosine before scoring. Approximal plaque (AP) were registered and expressed
separately.

Subgingival microflora
Sterile paper points were used to collect subgingival plaque samples from four sites in
each subject. Supragingival plaque was removed by a sterile cotton roll. Samples were
collected from the distobuccal aspects of all second molars (in case of missing second
molar, the first molar was used). The four samples were pooled in one Eppendorf tube
and kept at -20ºC until subsequent analysis. All samples were sent to the microbiology
laboratory at the University of Bern, Switzerland, for analysis by checkerboard DNA-
DNA hybridisation technique (Socransky et al., 1994; Socransky et al., 2004; Katsoulis
et al., 2005). The assay included a panel of 74 bacterial species (Baumgartner et al.,
2009).

Statistical analyses
The sample size calculation was based on a previous cross over study comparing
Miswak and toothbrush in 15 participants, in which a significant difference in PI and
GI between Miswak and toothbrush was found (Al-Otaibi et al., 2003). Based on this
study, the power calculation revealed a sample size of 25 in each group would have
80% power to detect a difference between groups in plaque index means of 0.19,
assuming that the common standard deviation is 0.23 using a two group t-test with a
0.05 two-sided significance level. The data were analysed using the Statistica (8.0)
program*: p” 0.05 was considered statistically significant. The results from the plaque
registrations were normally distributed (tested with Kolmogorow–Smirnow test), and
were expressed as mean and standard deviation. Differences were tested using
Student’s t-test. The gingival index results were not normally distributed and were
presented as median and quartile range and the differences were tested using Mann
Whitney U-test and Wilcoxon Signed rank test.

*
StatSof, Inc. Tulsa, Oklahoma, USA.

16
RESULTS

PAPER I

The pH of the 20% (w/w) freshly prepared aqueous Miswak extract ranged from 4.50
to 4.60. Titration of this extract with 0.02% HCl (pH 2.3) provided no evidence of a
buffering capacity of the Miswak extract. The Miswak extract did not keep the pH of
the mixture at any time point, as demonstrated in Figure 7.

All subjects had the classical plaque pH drop after 5% sucrose rinse. The maximum pH
drop was recorded after 8 min, with a mean minimum pH of 4.5 (95%CI 4.22 to 4.79):
the pH curve generated from one of the subjects is presented in Figure 8. Rinsing with
Miswak extract or water at 9 min raised the plaque pH immediately. However, Miswak
rinse maintained the elevated plaque pH level until the end of the session, whereas,
water rinsing did not. The difference in plaque pH between water and Miswak sessions
was statistically significant at 30 min (p< 0.001), 45 and 60 mins (p< 0.05) [Figure 9
and Table 1].

The difference in plaque pH between readings at baseline and each time point reading
was calculated for both Miswak and water. This change was most pronounced at 30
min. The mean (95% CI) change in the plaque pH was –0.5 (-0.8 to -0.2) and –0.9 (-1.2
to -0.5) for Miswak and water, respectively; this difference was statistically significant
(p< 0.001) [Figure 10].

The measurements also revealed that plaque pH values of the right side were lower
than those of the left side. The mean (95% CI) of the minimum readings of right and
left sides were 4.75 (4.47: 5.03) and 4.88 (4.62: 5.14) respectively, and was statistically
significant (p < 0.05) [Figure 11].

Miswak extract stimulated parotid saliva flow rate significantly (p < 0.01). The mean
unstimulated flow rate was 0.06 ± 0.03 (SD) ml/min and the stimulated rate was 0.2 ±
0.06ml/min. Stimulation was high during the 2 min rinse, then, it decreased
progressively during the subsequent period (0-5 min after stimulation).

Table 1: Mean (95% CI) and statistical significance of sucrose, Miswak, and water sessions at
different time points.

Plaque pH P-values for comparisons

Time
(min) Sucrose Miswak Sucrose
Sucrose Miswak Water
vs Miswak vs water vs water
13 5.47 (5.22 to 5.72) 6.03 (5.65 to 6.40) 5.63 (5.25 to 5.99) < 0.001 < 0.01 > 0.05
15 5.80 (5.46 to 6.14) 6.31 (5.91 to 6.70) 5.91 (5.43 to 6.39) < 0.01 > 0.05 > 0.05
30 5.74 (5.36 to 6.13) 6.11 (5.72 to 6.49) 5.57 (5.21 to 5.94) < 0.05 < 0.001 > 0.05
45 6.12 (5.57 to 6.68) 6.22 (5.88 to 6.57) 5.92 (5.59 to 6.24) > 0.05 < 0.05 > 0.05
60 6.34 (5.85 to 6.81) 6.44 (5.97 to 6.85) 6.08 (5.67 to 6.48) > 0.05 < 0.05 < 0.05

17
 Miswak pH= -0.0118(HCl in m l)+ 4.51
R 2= 0.9924
4.52

4.50

4.48

4.46

4.44
miswk pH

4.42

4.40

4.38

4.36

4.34

4.32

4.30
0 2 4 6 8 10 12 14 16

HCL in ml

Figure 7: The buffering capacity of Miswak water extract. The linear relation between the
added volume of acid and the resulting measured pH of the Miswak extract showing no
buffering capacity of the extract.

7.0
6.8
6.6

6.4
6.2
6.0

5.8
Plaque pH

5.6
5.4
5.2

5.0
4.8
4.6

4.4
Suc M
4.2
Miswak M
0 10 20 30 40 50 60
W at er M
T im e (m in)
Figure 8: A classic Stephan pH curves in the three sessions for one subject.

18
 7.0
**
6.8
**
6.6
*
6.4

6.2

6.0
plaque pH

5.8

5.6

5.4

5.2

5.0

4.8
Sucrose M
4.6 Miswak M
-10 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
Water M
Time
Figure 9: Means (95% CI) of plaque pH measurements during the Miswak, water, and no
rinse sessions. Statistically significant differences between the three test sessions are marked in
the figure (* p < 0.001; ** p < 0.05).

0.4

0.2

0.0
*
-0.2

-0.4
Chang plaque pH

-0.6

-0.8

-1.0

-1.2

-1.4

-1.6

-1.8 Change M
-5 0 5 10 15 20 25 30 35 40 45 50 55 60 65
Change W
Time (min)
Figure 10: Change in the plaque pH for the Miswak and water. Each point on the graph
represents the mean change and 95% CI for the 10 readings for water and Miswak sessions.

19
 6.8

6.6

6.4

6.2

6.0
Mean plaque pH

5.8

5.6

5.4

5.2

5.0

4.8

4.6
water R
0 1 2 3 4 5 6 7 8 9 10 11
water L
subject

Figure 11: The mean readings between right and left sides of water sessions. Each point
represents the plaque pH reading for each subject, showing right and left sides separately.

20
PAPER IV

Study Population
Fifty-eight of the original 68 participants completed the study. Of the 10 dropouts, two
men and three women were from the test group and five women from the control group
[Table 2].

Table 2: Number of subjects, gender, and mean age (range).

Group Subjects Gender (Male- Female) Mean age (range)

Test 30 16M – 14F 32.8 ( 18-54)
Control 28 3M – 25F 27.7 (18- 49)
M = male; F = female.

Plaque Index
In the test group, supragingival plaque decreased significantly (p=0.007) during the
three-week study, however, the decrease in the control group did not reach significance
(p=0.46). While the PI of the test and control groups was similar at baseline (p=0.18),
the difference at follow-up was significant (p=0.016). Both the test and control groups
showed intergroup significant decrease in AP (p=0.024 and p=0.003 respectively).
However; there was no significant difference in the change of PI and AP between the
two groups [Table 3].

Table 3: Mean and standard deviation of plaque index and approximal plaque at baseline and
follow-up.

Parameter Time point Test Control p-value (between

group group the two groups)

Plaque Index (PI) Baseline 2.27 ± 0.57 2.54 ± 0.89 0.18

Follow-up 2.00 ± 0.53 2.44 ± 0.78 0.016
p-value 0.007 0.46 NA
(between
time points)

Approximal plaque (AP) Baseline 2.62 ± 0.65 2.79 ± 0.89 0.39

Follow-up 2.30 ± 0.55 2.29 ± 0.84 0.95

p-value 0.024 0.003 NA

(between
time points)
Change in PI NA – 0.27 ± – 0.10 ± 0.71 0.31
0.51
Change in AP NA – 0.32 ± – 0.51 ± 0.83 0.36
0.72
NA = not applicable.

21
Gingival Index
In the test group, the GI decreased significantly by the end the three-week test period
(p=0.02), while no such reduction occurred in the control group (p=0.15). There was no
significant intergroup decrease in approximal GI in either group (p=0.46 for test group
and p=1.00 for control group). The change in GI and approximal GI did not differ
significantly between the two groups [Table 4].

Microbial analysis
The microbial analyses disclosed no significant changes between baseline and follow-
up samples in either group (data not shown).

Table 4: Median and quartile range of gingival index and approximal gingival index at
baseline and follow-up.

Parameter Time point Test Control p-value (between

Group group the two groups)
Gingival Index (GI) Baseline 1.07 (0.22) 1.00 (0.13) 0.10
Follow-up 1.04 (0.22) 1.00 (0.18) 0.11
p-value 0.02 0.15 NA
(between
time points)

Approximal GI Baseline 1.09 (0.16) 1.00 (0.29) 0.008

Follow-up 1.08 (0.23) 1.00 (0.19) 0.007
p-value 0.46 1.00 NA
(between
time points)
Change in GI NA – 0.03 (0.16) – 0.02 0.50
(0.15)
Change in Approximal GI NA – 0.02 (0.15) 0.00 (0.36) 0.98
NA = not applicable

22
DISCUSSION
The first study was a small clinical trial aiming to test a conventional crude Miswak
extract, a water extract used in several studies to test Miswak from different aspects.
Rinsing with Miswak extract had a neutralising effect on plaque pH after a previous
sucrose exposure. The data indicated rinsing with Miswak extract raised the plaque pH
for a more prolonged time as compared to water rinsing.

The pH of Miswak extract prepared in this study ranged from 4.50 to 4.60: this pH
value was consistent with previous studies (Almas et al., 1997; Almas, 1999).
Although, Almas (2001) reports the pH of Miswak extract was 5.7, the difference in the
pH of Miswak extract may be explained by differences in the methods of extract
preparation. In our study, the extract was prepared from fresh Miswak, whereas in
Almas’ study, Miswak sticks were dried for two days before preparation of the extract.
The time Miswak is kept before preparation also affects its pH: the pH of fresh Miswak
extract is 4.9 and the pH of one month-old Miswak is 5.5 (Almas et al., 1997).

The mechanism behind the elevated plaque pH with Miswak rinse could be due to a
buffering capacity of the Miswak extract, salivary stimulation due to Miswak taste,
and/or antibacterial activity against acid-producing bacteria. The primary laboratory
tests proved Miswak extract was acidic, and did not indicate any buffering ability when
titrated with 0.02% HCl. This suggested the rise in plaque pH was due to other reasons
[Figure 7]. Miswak stimulated salivary secretions as it has a relatively strong taste. It is
well known that salivary stimulation increases salivary flow rate washing out and
diluting acids (Edgar and O'Mullane, 1996). Detailed in vitro studies indicate (flow-
dependant) salivary film velocity is a more important factor in pH recovery of thinner
plaque, whereas, bicarbonate availability is more important for thicker plaque layers
(Macpherson et al., 1991). The effect of Miswak was most pronounced after 30 min
and remained up to 60 min suggesting an additional mechanism with increased salivary
flow. S. Persica has an antibacterial effect on different types of bacteria, including
mutans streptococci (Al-Lafi and Ababneh, 1995; Almas et al., 1997; Almas, 1999;
Almas and Al-Bagieh, 1999). However, information on the biologically active
compounds contributing to the reported antibacterial effects of S. Persica Miswak are
relatively limited (Wu et al., 2001). The growth and acid production of mutans
streptococci are inhibited by different types of substances such as Xylitol (Kakuta et al.,
2003; Miyasawa et al., 2003), and it is possible that Miswak may contain substances
that inhibit growth and acid production of mutans streptococci.

Earlier studies show that the consumption of food containing fermentable

carbohydrates causes rapid decrease in plaque pH, and recovery of low pH values is
rather slow. However, this recovery is detected differently, depending on the plaque pH
measuring method (Harper et al., 1985; Lingström et al., 1993a; Lingström et al.,
1993b; Imfeld et al., 1995). The micro-touch electrode method is effective and reliable
for assessing changes in plaque pH, and for identifying and ranking acidogenic foods
(Scheie et al., 1992; Lingström et al., 1993b). Plaque pH dropped after the 5% (w/w)
sucrose rinse, indicated good acid production by the subjects even though they

23
collected plaque for only three days before the start of the study. In the water session
[Figure 9], the plaque pH did not go back close to base pH value, even at the last two
time points (45 min and 60 min); whereas, at the end of the sucrose session, the plaque
pH reached a level close to baseline pH. It is possible that rinsing with water diluted the
buffering effect of saliva, whereas in the sucrose session the buffering effect of saliva
was maintained, as subjects did not have a second rinse after the sucrose rinse (Edgar
and O'Mullane, 1996).

The difference between right and left plaque pH readings was statistically significant in
the water session (p < 0.05); however, the difference was not statistically significant in
the Miswak and sucrose sessions. Earlier studies show that plaque pH is lower in the
upper jaw than in the lower and there are differences in plaque pH between different
sites in the mouth (Kleinberg and Jenkins, 1964; Lingström et al., 1993a). However a
difference between the right and left side has not been reported.

The difference found in this study could be explained by differences in the amount and
quality of plaque present on each side, although visual inspection did not indicate such
differences. Studies on the distribution of plaque in relation to recession and dentine
hypersensitivity indicate that in the upper arch, buccal plaque is more predominant on
all right-sided teeth that on left contra lateral teeth; corresponding to a predominantly
right-handed toothbrushing population. This means that right-handed subjects brush
their upper left side better than their upper right side (Addy et al., 1987a; Addy et al.,
1987b; Rees, 2000; Addy and Hunter, 2003). Professional tooth cleaning before the
start of the study could have resulted in no difference between right and left readings;
although, more than three days "of no brushing" may be needed to allow for sufficient
plaque accumulation for acid production to be detected. However, none of the plaque
pH studies has included professional teeth cleaning before plaque pH measurement.

As the crude Miswak extract was tested in the first study (Paper I), it was necessary to
test Miswak as it is used in real life. It was also important to compare Miswak with
something that has a similar mechanical plaque removing technique, the idea of
deactivating Miswak and using it as a control came into action. In this thesis, the effect
of Miswak on plaque and gingival indexes was tested. Clinical studies comparing adult
habitual Miswak users and habitual toothbrush users reveal better periodontal status in
Miswak users (Gazi et al., 1990; Darout et al., 2000a). Moreover, it has been proven
that the chewing stick is as or more effective than the toothbrush in reducing plaque
and gingivitis (Olsson, 1978; Danielsen et al., 1989; Gazi et al., 1990; Al-Otaibi et al.,
2003).The findings from the present study supported previous findings about Miswak.

In order to evaluate a possible chemical effect of the Miswak, approximal plaque (AP)
was analysed separately and both fresh and deactivated Miswak reduced AP
significantly, which is also emphasised in the results of another study comparing the
effectiveness of Miswak and toothbrush on proximal plaque control (Al-Otaibi et al.,
2003). However, in the present study, intergroup comparison disclosed greater
reduction of AP in the control group than in the test group. This suggested a limited
chemical effect of fresh Miswak in areas difficult to reach, as is mentioned by Gazi et al
(1990). This difference might be attributable to the fact that deactivated Miswaks used
as a control lacked their natural moisture and content. Thus, the participants in the

24
control group may have needed to be more assiduous in mechanical cleaning to attain
the level of cleanliness they were accustomed to achieving with routine Miswak use. In
order to isolate the chemical effects of Miswak from the mechanical effects, it would
probably be necessary to use Miswak extract of some kind rather than the sticks per se.

Darout et al. (2003) use the checkerboard hybridisation technique to compare

subgingival plaque samples of regular miswak and toothbrush users, and report higher
A. actinomycetemcomitans in the plaque of regular miswak users. In a clinical,
controlled, cross-over study, Al-Otaibi et al. (2004), use the same technique to compare
subgingival plaque samples of Miswak and toothbrush groups and report significantly
lower A. actinomycetemcomitans in the Miswak group. However, the present study
disclosed no differences in subgingival microbiota for any of the bacterial species
tested. One explanation might be that all the participants of the present study were
regular Miswak users and this had already influenced their subgingival microbiota.
Consequently, the two-week wash-out period might have been too short for eliminating
the effect of previous Miswak use. A different result might have been achieved if the
participants selected for the study had never used Miswak before.

The participants of the present study had gingivitis with no signs of periodontitis.
Theilade et al. (1966) describe the sequential development of gingival plaque, from a
simple monolayer of Gram-positive coccoid bacteria colonising the enamel surface and
the marginal gingiva to a complex microbial plaque dominated by Gram-negative
anaerobic cocci, filaments, and spirochetes. However, the present studies indicate that
Miswak had higher antibacterial effect on Gram- negative bacteria. Moreover, Miswak
essential oil extract tested on seven Gram-positive and seven Gram-negative bacterial
species (Paper III), indicated that Miswak extract was more effective against Gram-
negative than Gram-positive species. Thus, a corresponding study on patients with
periodontitis, with predominant Gram-negative microflora, might have disclosed
clinically relevant differences in the subgingival microflora.

The active and deactivated Miswak groups had more female than male participants.
Testing for an influence of the differences in gender on the data did not reveal any
significant effect on the study results. Moreover, the number of dropouts did not have
any effect on the results as it was equal in both groups. The reasons for not continuing
participation are unrelated to the study.

25
 LABORATORY PART (PAPERS II & III)
Miswak water extract had an effect on the plaque pH, and brushing with fresh Miswak
reduced dental plaque and gingival inflammation (Papers I & IV). This raised the
question of how did Miswak really affect oral bacteria and whether laboratory testing
of Miswak as it is used clinically, with out extraction, would have any effect on oral
pathogens. As Miswak displayed a stronger antibacterial activity against the oral
pathogens (Paper II) than previous reports on Miswak extracts, the need to have an
extract that represented the same strong antibacterial activity as was achieved with
fresh Miswak was raised. Extraction of Miswak by steam distillation and by Medium
Pressure Liquid Chromatography produced an extract with the strong antibacterial
activity required. This lead to the necessity of chemically characterising the
components of the Miswak sticks and to defining if one or more of these components
was the active component (Paper III).

MATERIALS AND METHODS

PAPER II

Miswak pieces preparation

Standardised Miswak pieces were prepared with sticks 0.5 mm wide. The sticks were
cut into pieces weighing 0.14, 0.07, 0.03, or 0.015 g. The larger pieces (0.14g and
0.07g) were tested against all bacteria used in this study, while the smaller pieces
(0.03g and 0.015g) were only tested against P. gingivalis. The outer cork layer of the
Miswak [Figure 12] was removed just before weighing and testing.

Bacterial strains and cultivation

The bacterial strains selected for antimicrobial testing of the Miswak were: S. mutans
(CCUG 27624; Ing-Britt), L. acidophilus (NCTC 1723), A. actinomycetemcomitans
(HK 1519), a highly leukotoxic strain*, P. gingivalis (ATCC 33277), and H. influenzae
(ATCC 49247).
S. mutans and L. acidophilus were grown for two days under anaerobic conditions on
Colombia base agar† supplemented with 0.01% tryptophan‡ and citrated horse blood
(5%). A. actinomycetemcomitans was grown for two days on the same medium, but
incubated in 95% air and 5% carbon dioxide. P. gingivalis was grown anaerobically for
four days on Colombia base agar supplemented with hemin (0.05 mg/ml), vitamin K
(0.01 mg/ml), and citrated horse blood (5%). H. influenzae was grown for one day in
95% air and 5% carbon dioxide on haematin-agar§ supplemented with haemoglobin
(1%) and isovitalex (1%).

*
Dr M. Kilians strain collection, Aarhus, Denmark.
†
Acumedia, Baltimore, MD, USA.
‡
Merck, WWR International AB, Stockholm, Sweden.
§
GC-agar, Acumedia, Baltimore, MD, USA.

26
Figure 12: A cross section of the Miswak showing the different layers of the root: (a) Cork,
(b) Cortex, (c) Phloem, (d) Xylem, (e) Pith.

Preparation of bacterial suspensions

The suspension of S. mutans and L. acidophilus was prepared in PBS (Phosphate
Buffer Solution). A. actinomycetemcomitans and H. influenzae were suspended in
HTM broth (Haemophilus Test Medium, according to CLSI document M7-A7, 2006).
P. gingivalis was suspended in PYG (Peptone Yeast Glucose media, prepared
anaerobically). The turbidity of all suspensions was standardised for each bacterial
strain using a spectrophotometer*. The suspensions were adjusted to give § 3×108 CFU/
ml. Each bacterial suspension was then swabbed over the surface of the special agar
plate.

Antibacterial testing

Two types of tests were conducted

In the first test, a standardised round hole, 5 mm in diameter, was punched into the
middle of each inoculated agar plate, and a 0.14 g Miswak piece was placed in the hole.
This test was repeated ten times for each strain.
The second test was designed to determine the presence of volatile, air-borne
antibacterial compounds. A 0.14 g Miswak piece was suspended by a 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. In both tests, two plate sizes, 8 and 14 cm, were used, depending on the
strength of the antibacterial effect of the Miswak piece on the bacterial strain. In
addition, in the second test lower weights of Miswak pieces were tested in duplicate.

Statistical analyses
The data were not normally distributed and non-parametrical statistical tests were used.
The Mann-Whitney U-test was used to compare the inhibitory effects of 0.14 g Miswak

*
Ultrospec® 3000, Cambridge, England.

27
samples embedded in inoculated agar plates and samples suspended above the plates. It
was also used to compare the inhibitory effects of 0.14 g and 0.07 g Miswak samples
suspended above the inoculated agar plates. The Kruskal-Wallis test was used to
compare the inhibitory effects on P. gingivalis of four different weights of suspended
Miswak. The data were analysed using the STATISTICA (7.0) program*. P<0.05 was
considered statistically significant.

PAPER III

Laboratory work was in four parts: Miswak extracts preparations, Miswak extracts
chemical analysis, antibacterial testing of the Miswak extracts, and transmission
electron microscopy.
I. Miswak extracts preparations
1) Miswak oil prepared by steam distillation.
2) Combined extraction-fractionation of fresh Miswak using Medium Pressure
Liquid Chromatography (MPLC).
II. Chemical analyses of Miswak extracts
1) Gas Chromatography-Mass Spectrometry (GC-MS) analysis of Miswak oil and
Miswak fractions obtained from MPLC.
2) Analysis of volatiles from fresh Miswak using Solid phase microextraction
(SPME).
III. Antibacterial testing of the Miswak extracts
1) Growth conditions and bacterial strains selected for antimicrobial testing of the
Miswak extracts.
2) Testing Miswak oil.
3) Testing Miswak pooled fractions.
IV. Transmission electron microscopy.

I. Miswak extracts preparations

1) Miswak oil preparation by steam distillation

Many organic compounds tend to decompose at high sustained temperatures. The test
for deactivation of Miswak indicated Miswak loses its antibacterial activity on heating;
thus, separation by normal distillation was not an option. Therefore, steam distillation
was chosen for obtaining the Miswak oil. The addition of water or steam depresses the
boiling points of the compounds, allowing the compounds to evaporate at lower
temperatures, preferably below the temperatures at which deterioration of the material
becomes detectable. Miswak sticks (1.5 Kg) were cut into pieces of 1- 2 cm in length.
The pieces were then smashed with a stone mill, place into a 5 litre round-bottom flask
[Figure 13], then, 700 ml of distilled water was added and the mixture was heated. The
flask was fitted with a thermometer and a condenser. The temperature was maintained
at 80°C. The steam from the heated flask was condensed in the condenser with cold tap
water [Figure 14]. The result was Miswak oil mixed with water. The miswak oil was
separated from the water by extracting the oil/water mixture with hexane †. The hexane
was dried with magnesium sulphate (Mg2So4), and then evaporated under pressure

*
StatSof, Inc. Tulsa, Oklahoma, USA.
†
Merck©,Val-De-Reuil Cedex, France.

28
using the Rotavapor® *[Figure 16, C]. This procedure resulted in 12 grams of pure
Miswak oil.

Figure 13: A) Miswak roots cut into small pieces. B) Miswak root smashed with a stone mill.
C) Miswak root pieces in round bottomed flask.

Figure 14: Steam distillation set-up.

2) Combined extraction-fractionation of fresh Miswak using Medium Pressure Liquid

Chromatography (MPLC)
The preparative liquid chromatography technique used in this study was the same as
described by Liblikas et al. (2005). Fresh Miswak was cut into 1-2 cm pieces and

*
Rotavapor® R-210/ R215, BÜCHI Labortechnik AG, Flawil, Switzerland.

29
ground with silica gel* in a 1:1 mixture (50.4 g Silica + 50.4 g Miswak) by means of a
household meat grinder. The material was passed through the grinder four times to
produce a free flowing powder [Figure 15]. The Miswak silica powder (100.8g) was
dry packed in a 30 mm internal diameter glass column followed by fresh silica gel (18.4
g). The length of the loading zone was 19 cm and that of the fresh silica was 5.6 cm.
The column was sealed with pistons that could be adjusted to the resulting bed length
[Figure 16, A].

Figure 15: A) Miswak pieces, B) Fresh silica, C) Ground Miswak with silica.

Figure 16: A) The column with Miswak and silica: 1. Silica & sample (19 cm), 2. Pure silica
(5.6 cm), where separation takes place, B) fractions eluted from the column in A, C) The
Rotavapor® used to reduce the amount of solvents.

The combined extraction and chromatography was performed with the setup depicted
in Figure 17. Two consecutive gradients were used. The first gradient was attained with
100 ml of 100% hexane in the mixing chamber followed by consecutively adding 100
ml of mixtures of hexane and ethyl acetate (EtOAc) (1.25, 2.5, 5, 10, 20, 40, and 80),
ending with 300ml of 100% EtOAc to the solvent reservoir. This was followed by

*
Merck 60, 230-400 mesh (0.040- 0.63 mm).

30
consecutively adding 100 ml of mixtures of methanol (MeOH) in EtOAc (1.25, 5, 10,
20, 40, and 80), ending with 300ml of 100% MeOH. The eluent was collected in 25 ml
test tubes [Figure 16, B].

All fractions were checked with thin layer chromatography (TLC) performed on silica
gel plates (Merck 60, pre-coated aluminum foil) and eluted with: 10% EtOAc in hexane
for fractions 1-25; 20% EtOAc in hexane for fractions 20-41; or, 30% MeOH in ethyl
acetate for fractions 40-71. The plates were then sprayed with a solution containing 3 g
vanillin dissolved in ethanol (EtOH) 99% with 0.5% sulphuric acid, and developed by
heating the TLC plates [Figure 19]. The MPLC fractions were pooled guided by the
TLC results; fractions containing compounds with similar Retardation factor (Rf)
values. This Resulted in eight pooled fractions [Figure 18,Table 5]. The solvents in the
eight pooled fractions were evaporated using Rotavapor®. The pooled fractions for the
antibacterial tests were recollected by dissolving a defined amount in dimethyl
sulfoxide (DMSO). For GC-MS analyses, pooled fractions were recollected in
Dichloromethane (DMC), except sample seven that was recollected in 50%
DMC/EtOH.

Figure 17: Describing the setup for chromatography. 1. Column; 2. Quick Grip Vise; 2 b.
Column holder; 2 c. Stainless steel mounting device; 3. Airtight mixing chamber; 4. Luerlock
connectors; 5. Pump; 6. Magnetic stirrer; 7. Solvent reservoir; 8. Stock solutions for gradient
elutent; 9. Fraction collector; 10. UV-detector; 11. Chart recorder. (Baeckström)

Figure 18: TLC for pooled MPLC fractions (1-6), resulting in fractions eluted in 10% ethyl
acetate in hexane.

31
Figure 19: A) MPLC fractions 1-25 eluted with 10% ethyl acetate in hexane, B) Fractions 20-
41 eluted with 20% ethyl acetate in hexane, C) Fractions 40-71 eluted with 30% methanol in
ethyl acetate.

32
Table 5: The pooled MPLC fractions gave 8 pooled fractions, their Retardation factor
and amount in grams.

Pooled Rf Tube Fraction Pooled Rf Tube Fraction

Fractions number weight Fractions number weight
1 1.0 1-7 0.014 g 5 0.2 24-30 0.044 g
2 0.4 8-18 0.234 g 6 0.1 31-53 0.08 g
3 0.5 &0.4 19-20 0.029 g 7 > 0.1 54-56 0.117 g
4 0.4 &0.3 21-23 0.039 g 8 > 0.1 57-70 2.721 g
Rf = Retardation factor

II. Qualitative and Quantitative Chemical analyses for Miswak extracts

1) GC-MS analysis of Miswak oil (from steam distillation) and Miswak pooled
fractions from MPLC
Gas chromatography-mass spectrometry (GC-MS) was performed using a Varian 3400*
GC connected to a Finnigan SSQ 7000 quadropole mass spectrometer†. The GC was
equipped with a split/splitless injector (splitless mode 30 sec), and a DB-wax capillary
column‡ (30 m, 0.25 mm id, and 25 μm film thickness). Injector temperature was
isothermally set at 230°C. The carrier gas (He) was of high purity and delivered at a
constant pressure of 10 bars. The temperature programme was 40°C for 1 minute,
followed by an increase in temperature of 4°C /min up to 235°C: the temperature was
then maintained at 235°C for 10 minutes. The transfer line temperature was kept at
240°C. The filament off time was 4 min and the MS ion source temperature was 150°C.
Mass spectra were obtained at 70 eV with a mass range of 30 to 600 m/z.

All MPLC pooled fractions and oil samples for GC-MS analysis were prepared in
hexane by dissolving 10 mg of each completely dried pooled fraction in 1 ml of
hexane; a 100 ȝl ethyl acetate were added to increase solubility. The pooled fractions
were further diluted to 2mg/ml by addition of hexane, of which 1 ȝl was injected for the
analysis: pooled fractions 7 and 8 were not run on GC-MS for technical reasons. For
analysis of the volatiles from the cut Miswak sticks, the SPME fiber was desorbed in
the injector for 4 minutes. The constituents of the pooled fractions were identified by
comparison with the Finnigan of the national institute of standards and technology
library (NIST) and confirmed by analysing the reference compounds using the same
chromatographic parameters.

For quantitative determination of the main constituent (benzyl isothiocyanate) in oil

distillate and pooled MPLC-fractions, five different concentrations of a synthetic
reference of benzyl isothiocyanate (BITC)§ were injected into the GC-MS under the

*
VARIAN 3400 Chromatography system, Walnut Creek, California, USA.
†
SSQ 7000 Finnigan Mat, Thermo Scientific system Inc, California, USA.
‡
J & W Scientific, Folsom, California, USA.
§
>98% GC-purity, Lancaster Synthesis Inc., Windham, NH, USA.

33
same conditions as for the pooled fractions and oil dilutions. A calibration curve was
obtained with Y = 4.7495E7 + 1.7387E9*x and R2= 0.9953 [Figure 20].

2) Analysis of volatiles from fresh Miswak using Solid phase microextraction

(SPME):
A piece of Miswak stick (20 mm) from the roots of Salvadora persica was placed in a 5
ml beaker. The volatiles from the cut root were collected for 15 minutes on a solid-
phase microextraction (SPME) fiber coated with the polymer
polydimethylsiloxane/divinylbenzene*. The volatiles were analysed by GC-MS, see
above.

Figure 20: The calibration curve of synthetic Benzyl isothiocyanate generated from five
different concentrations. The curve formula is: y = 4.7495E7 + 1.7387E9*x; p = 0.0001; R2 =
0.9953.

III. Antibacterial testing of the Miswak extracts

1) Growth conditions and bacterial strains selected for antimicrobial testing of the
Miswak extracts
Gram-negative bacteria: Aggregatibacter actinomycetemcomitans HK 1519†,
Haemophilus influenzae (ATCC 49247), Escherichia coli strain MC4100, Escherichia
coli strain D21 (9), Salmonella enterica serovar Typhimurium (ATCC 14028),
Pseudomonas aeruginosa strain PA01, and Gram-negative bacteriodetes
Porphyromonas gingivalis (ATCC 33277).

Gram-positive bacteria: Streptococcus mutans (CCUG 27624), Lactobacillus

acidophilus (NCTC 1723), Streptococcus pyogenes clinical isolate‡, Staphylococcus

*
Supelco, Sigma-Aldrich, Sweden.
†
Dr M. Kilians, Aarhus, Denmark.
‡
Dr Martin Lindberg, Uppsala, Sweden.

34
aureus strain 8325-4*, Lactobacillus fermentum (DSM 20052), Enterococcus faecalis
(ATCC 29212), and Enterococcus faecium, clinical isolate†

Growth conditions: A. actinomycetemcomitans was propagated for two days on

Colombia base agar‡ supplemented with 0.01% tryptophan§, and citrated horse blood
(5%) in 5% CO2 gas-pack generated atmosphere**. P. gingivalis was propagated for six
days on Colombia base agar supplemented with hemin (0.05 mg/ml), vitamin K (0.01
mg/ml), and citrated horse blood (5%) in anaerobic atmosphere generated with gas-
packs††. For the antibacterial assays, colonies of A. actinomycetemcomitans and P.
gingivalis were suspended in Peptone Yeast Glucose medium (PYG) to an optical
density of 0.5 at O=590 nm. S. mutans was grown on BHI‡‡ (Brain Heart Infusion) agar
plates for two days in 5% CO2 atmosphere generated with gas-packs. For antibacterial
assay, colonies were suspended in BHI broth (Oxoid)) to the same optical density as
above. H. influenzae was propagated over-night on GC-agar§§ supplemented with
haemoglobin (1%) and isovitalex (1%) in 5% CO2 atmosphere generated with gas-
packs. For the antibacterial assay, bacteria colonies were suspended in 5% CO2
preconditioned BHI medium with 5% Fildes Enrichment (BBL™) to the same density
as above. L. acidophilus and L. fermentum were propagated for two days on
Lactobacilli MRS agar plates*** in 5% CO2. For antibacterial assay, colonies were
suspended in Lactobacilli MRS Broth to the same optical density as above.
S. pyogenes, S. aureus, E. faecalis, and E. faecium were all propagated over night on
TSB agar plates††† in normal atmosphere, supplemented with 0.5% yeast extract
(BBL™). The bacteria were suspended in TSB with 0.5% yeast extract as above for the
antibacterial assay. E. coli MC4100, E. coli D21, P. aeruginosa, and S. typhimurium
were propagated over night in normal atmosphere on Luria Agar Base, Miller
(Difco™). For the antibacterial assay, the bacteria were grown in Luria Broth (Difco™)
to the same optical density as above.

2) Testing Miswak oil

Dose-response analysis of S. persica essential oil and pure synthetic BITC

Miswak oil was tested for antibacterial activity in six dilutions and one pure oil sample.
Bacteria were incubated for 90 minutes in respective growth medium at 37 ºC in the
presence of different concentrations of essential oil. Dilutions of oil was prepared in

*
Dr S Arvidsson, Karolinska Institutet,Stockholm, Sweden.
†
Inger Kühn, Karolinska Institutet, Stockholm, Sweden.
‡
Acumedia, Baltimore, MD, USA.
§
Merck, WWR International, Sweden.
**
BBL™, CampyPak™, Becton Dickinson, Sweden.
††
GasPak™, Becton Dickinson, Sweden.
‡‡
Oxoid, Malmö, Sweden.
§§
Acumedia, Baltimore, MD, USA.
***
Difco™, Becton Dickinson, Sweden.
†††
Tryptic Soy Broth and Bacteriological agar No 1, Bacto™, Becton Dickinson, Sweden.

35
DMSO* and 5 Pl of each dilution or undiluted oil was added to 500 Pl bacteria
suspension to obtain final dilutions of 1%, 0.1% 0.05%, 0.02%, 0.01%, 0.005%, and
0.001% oil. These oil dilutions corresponded to 28Pmole, 2.8 Pmole, 1.4Pmole, 0.56
Pmole, 0.28 Pmole, 0.14Pmole and 0.028 Pmole pure BITC respectively. Only 5Pl of
DMSO was added to the controls. After incubation, the bacteria were plated on
respective agar-medium plates for determination of live bacteria by counting the
number colony forming units (CFU). Each experiment was in duplicate and every
experiment was repeated three times.

Pure synthetic Benzyl isothiocyanate (BITC) with a dilution of 1%, 0.1%, 0.05%,
0.02%, 0.01%, 0.005%, and 0.001% was used for the dose response test. The test was
performed on E. coli MC 4100 and A. actinomycetemcomitans to compare its effect to
corresponding dilutions of S. persica oil on the same bacterial strains.

Killing kinetics of the essential oil

To test the kinetics of the Miswak oil, one of the dilutions was used on two Gram
negative bacteria. The number of viable A. actinomycetemcomitans and E. coli MC
4100 was assessed after co-incubation with essential oil at a final dilution of 0.1% and
0.02%. Aliquots were removed at 2, 5, 10, 20, 40, and 90 minutes, and plated on
respective agar-medium plates for determination of colony forming units. The
experiment was in duplicate and repeated three times.

Bactericidal effect of essential oil with increasing ratio of bacteria to oil

In this test the number of bacteria in the suspension tested was increased against fixed
amounts and dilutions of Miswak oil. The tests were with 0.1% essential oil on A.
actinomycetemcomitans and 0.05% essential oil on E. coli MC4100. Bacteria were each
incubated for 90 minutes in growth medium at 37 ºC with the respective oil dilution.
Five different densities with 10 times increasing steps of bacteria were assessed: 104,
105, 106, and 107 bacteria/ml. After incubation, the bacteria were spread on agar-
medium plates for determination of live bacteria by number of colony forming units
(CFU). Each experiment was in duplicate and every experiment was repeated three
times.

3) Testing the eight pooled fractions from MPLC

Miswak fractionation gave 8 pooled fractions that were tested on two Gram negative
bacteria E. coli MC4100 and A. actinomycetemcomitans and two gram positive bacteria
L. acidophilus (NCTC 1723) and S. pyogenes clinical isolate. Five Pl of each pooled
fraction were added to 500 Pl of bacterial suspension. Bacterial suspensions were
incubated for 90 minutes in respective growth medium at 37 ºC. To test synergistic
effect, all 8 pooled fractions were added together and five Pl were tested on each
bacteria. After incubation, the bacteria were plated on the respective agar-medium
plates for determination of live bacteria by counting the number of colony forming

*
D2650, Sigma-Aldrich, Stockholm, Sweden.

36
units (CFU). Each experiment was in triplicate and every experiment was repeated two
times.

IV. Transmission electron microscopy

In order to understand the nature of the fast killing exerted by the essential oil on Gram
negative bacteria, electron microscopy analysis was performed. The susceptible
bacterium A. actinomycetemcomitans was chosen to test the essential oil and to
compare that with BITC and ampicillin antibiotic. For control of essential oil and BITC
one sample was tested by adding 1% DMSO, and for the control of ampicillin 1%
water was tested. A. actinomycetemcomitans was incubated for 2, 20, or 40 minutes in
37 ºC with 0.1 % of essential oil (corresponding to 2.8 Pmole BITC). To determine if
there was a similar effect of BITC, the same test was run on 2.8Pmole of synthetic
BITC. For comparison 1mg/ml of ampicillin antibiotic* was tested. At each time point
an aliquot of bacteria suspension was removed and centrifuged for 10 min. The
supernatant was discarded and bacteria were suspended over night in fixation buffer
containing 2% glutaraldehyde in 0.1M sodiumcacodylate buffer with 0.1M sucrose and
3mM CaCl2, pH 7.4 at 4ºC. After fixation the bacteria were centrifuged to a pellet. The
pellet was rinsed in 0.1 M phosphate buffer (pH 7.4) followed by postfixation in 2%
osmium tetroxide in 0.1 M phosphate buffer (pH 7.4), at 4ºC for 2 h, dehydrated in
ethanol followed by acetone and embedded in LX-112 †. Sections were contrasted with
uranyl acetate followed by lead citrate and then examined in a Leo 906 transmission
electron microscope‡ at 80 kV(Laane et al., 2009). Digital images were taken using a
Morada digital camera§.

*
Sigma-Aldrich.
†
Ladd, Burlington, Vermont, USA.
‡
Zeiss, Oberkochen, Germany.
§
Soft Imaging System, GmbH, Münster, Germany.

37
RESULTS

PAPER II

The Miswak had powerful inhibitory effects on the growth of A.

actinomycetemcomitans, P. gingivalis, and H. influenzae, less effect on S. mutans, and
much less effect on L. acidophilus. The 0.14 g suspended Miswak exhibited
significantly greater inhibition on A. actinomycetemcomitans and H. influenzae than the
Miswak embedded in agar (P< 0.01, and P< 0.001 respectively). The 0.14 g suspended
Miswak was less effective than the corresponding embedded Miswak on S. mutans (P<
0.001), and had no inhibitory effect on L. acidophilus [Table 6].

Table 6: Growth inhibition (cm) of different bacteria with various Miswak weights.

0.14-g Miswak P-value Suspended Miswak

Bacterial strain in Agar Embedded & 0.14g (median 0.07g (median
(median [range]) Suspended [range]) P-value [range])
Miswak
A. actinomycetemcomitans 10.9 (10.0-14.0) 0.008 13.0 (12.0-14.0) 0.021 8.0 (7.8-8.2)

P. gingivalis 14.0 (11.0-14.0) 0.370 14.0 (14.0-14.0) 0.008 5.0 (5.0-5.0)

S. mutans 3.2 (2.6-4.6) 0.001* 0.46 (0.28) 0.021* No inhibition
L. acidophilus 1.4 (1.2-1.6) 0.007 No inhibition NA Not tested
H. influenzae 9.3 (5.1-11.5) 0.001 12.0 (11.5-13.0) 0.008 8.8 (7.2-9.0)
NA = not applicable. * Inhibition was less when Miswak was suspended.

The 0.07g suspended Miswak pieces exhibited significantly less inhibition on A.

actinomycetemcomitans and H. influenzae than the 0.14 g suspended piece (P< 0.05),
and no observable effect on S. mutans. The strain most sensitive to the Miswak piece
was P. gingivalis and least sensitive was L. acidophilus. The overall differences among
inhibition zones around P. gingivalis associated with Miswak pieces of different
weights were significant (P= 0.011).

Figure 21 shows the dose response of different suspended Miswak weights (0.14g,
0.07g, 0.03g, and 0.015g) on P. gingivalis. Examples of inhibition zones associated
with different methods of testing the Miswak are presented in Figure 22, which also
shows the testing of different Miswak weights and full growth of bacteria in the
absence of Miswak.

38
 Mean inhibition (cm)
16
14
14

12

10

6 5

2 1.5
0
0
0.14 0.07 1 0.03 0.015 Weight (g)

Figure 21: Correlation of P. gingivalis growth inhibition to Miswak of various weights

suspended with a thread 3mm above the agar plate.

Figure 22: Miswak-induced growth inhibition, all with 14 cm plates. (A) P. gingivalis growth
in the absence of Miswak. (B) P. gingivalis inhibition with 0.14g Miswak piece embedded in
agar. (C) P. gingivalis inhibition with 0.14g suspended Miswak. (D) P. gingivalis inhibition
with 0.07g suspended Miswak. (E) A. actinomycetemcomitans inhibition with 0.14g suspended
Miswak. (F) H. influenzae with 0.07g suspended Miswak.

39
PAPER III

The steam distillation produced 12 g of volatile essential oil, and that MPLC gave 8
pooled fractions. The next step was analysis of both oil and samples using GC-MS to
characterise and quantify the components of these two extracts.

Chemical analyses of Miswak extracts

GC-MS and SPME analyses of Miswak extracts

The GC-MS analysis for identification of Miswak oil optioned by steam distillation
gave: 69.6% Benzyl isothiocyanate (BITC) and 29.5% Benzyl nitrile (BN, benzyl
cyanide), with retention times of 35.6 and 31.2 minutes respectively [Figure 24].
The antibacterial testing of Miswak eight pooled fractions obtained by MPLC revealed
that the pooled fractions two, three, and five were the most effective against bacteria.
Identification analysis of these fractions with GC-MS revealed that fraction two was
100% BITC with a retention time of 35.6 min, fraction three was 99.2% BITC with
retention time of 35.6 min; and fraction five was 66.1% BITC, and 29.4% Benzyl
nitrile (Benzyl cyanide), with retention times of 35.6 and 31.2 minutes respectively.

The GC of all three active pooled fractions is presented in Figure 25. The MS for the
compound presented in this GC: compound A (Benzyl isothiocyanate) and compound
B (Benzyl nitrile) are presented in Figure 24 (II) and (III) respectively.
The SPME analysis of 15 minutes collection of volatiles from Miswak stick gave 98%
benzyl isothiocyanate with retention time of 28.9 min [Figure 23]. The MS of BITC is
shown in Figure 24 (II).

Figure 23: GC-MS chromatogram of Miswak volatiles showing BITC compound (A) as the
main component.

40
 I

II

III

Figure 24: Gas chromatography-mass spectrometry (GC-MS) analysis of S. persica essential

oil. I) Shows GC-MS of S. persica essential oil with two main compounds, II) MS and
chemical structure of compound (A) Benzyl isothiocyanate. III) MS and chemical structure of
compound (B) Benzyl nitrile.

41
 I

II

III

Figure 25: Gas chromatography (GC) of active antibacterial pooled fractions from MPLC.
I) GC-MS for pooled fraction no. two with only one compound (BITC). II) GC-MS for pooled
fraction no. three with only one compound (BITC). III) GC-MS for pooled fraction no. five
with two compounds (BITC and BN).

42
The major constituent of Miswak essential oil, the active fractions, and the volatiles of
fresh Miswak was Benzyl isothiocyanate. Using the formula from the calibration curve
of the synthetic reference of BITC, the quantity (the % age) of BITC present in Miswak
essential oil and each pooled fraction was calculated [Table 7]. The amount of BITC
added to the bacteria cultures, considering that BITC constitutes 73.8% of the oil
[Table 3], was 28μmole (1% dilution), 2.8 μmole (0.1%), 1.4ҏμmole (0.05%), 0.56
μmole (0.02%), 0.28 μmole (0.01%), 0.14ҏμmole (0.005%), and 0.028 μmole (0.001%)
for the different oil dilutions used. Synthetic BITC, used at the same concentration as it
occurs in the oil, was almost equally efficient against the oil-susceptible bacteria A.
actinomycetemcomitans and E. coli [Figure 26].

Table 7: Percentage of Benzyl isothiocyanate in essential oil and 1-6 pooled fractions from
MPLC

Pooled Pooled
fraction Benzyl isothiocyanate fraction Benzyl isothiocyanate
1 6.6% 5 22.9%
2 89.4% 6 6.1%
3 72.2% Essential Oil 73.8%
4 5.2%

E. coli MC4100 A. actinomycetemcomitans

10 4 10 4
10 3 10 3
CFU

CFU

10 2 10 2
10 1 10 1
10 0 10 0
l

0. 6
0. 8
0. 14
8
28
8
0. 4

l
28
8
0. 4
0. 6
28
0. 1 4
8
ro

ro
02
5
2
2.
1.

2.
1.
5

02
nt

0.
nt
Co

Co

BITC (Pmole) BITC (Pmole)

Figure 26: Dose-response analysis of antibacterial activity of synthetic BITC. The different
amount of BITC applied corresponds to the BITC content in the different oil dilutions of Figure
27.

43
Antibacterial testing of Miswak extracts

Dose response to S. persica Miswak essential oil and pure BITC

Miswak oil with different dilutions (1%, 0.1%, 0.05%, 0.02%, 0.01%, 0.005%, 0.001%
oil) had a strong antibacterial activity. The activity varied with different dilutions and
different bacterial strains.
In general, Gram negative bacteria were more sensitive to all oil dilutions than Gram
positive. The most sensitive Gram negative bacteria were P. gingivalis, and the most
resistant were P. aeruginosa. Miswak oil with 0.05% was very effective against most
Gram negative bacteria tested in this study. P. gingivalis and S. typhimurium had no
growth up to 0.02% oil dilution [Figure 27, A].

All Gram positive bacteria tested in this study were to some extent resistant to the oil. It
can be seen from the pattern of growth of all Gram positive bacteria that they were
affected, but not to the extent of the dramatic growth inhibition seen for Gram negative
bacteria. Only S. aureus displayed no bacterial growth at 1% oil dilution; however, with
the rest of dilutions tested, S. aureus had close to normal growth [Figure 27, B]. Pure
BITC had the same antibacterial power as the S. persica essential oil extract. It can be
observed that the killing pattern of BITC on E. coli MC 4100 and A.
actinomycetemcomitans was comparable to that of the essential oil [Figure 26].

Killing kinetics of the essential oil

In this section bacterial growths with two different dilutions tested at different time
points are presented.
A. actinomycetemcomitans: 0.02% Miswak oil reduced the viable bacterial number
from 1x104 to 1x10 by the end of the session, whereas, 0.1% Miswak oil reduced the
viable bacterial count from 1x104 to almost zero viable bacteria in 10 minutes, ending
with a completely sterile culture.

E-coli MC4100: 0.02% Miswak oil reduced the viable bacterial count from 1x104 to
almost zero in two minutes but ended with 1x10, whereas, 0.1% Miswak oil reduced
the bacterial growth from 1x104 to completely sterile culture in 5 minutes [Figure 28].

44
 E. coli MC4100 E. coli D21 S. typhimurium
10 4 10 4 10 4
10 3 10 3 10 3
CFU

CFU

CFU
10 2 10 2 10 2
10 1 10 1 10 1
10 0 10 0 10 0
l

l
1
0. 1
0. 5
02
0 . 01

1
0 . 05

1
05

0. 2
0 . 01
0. 5
1

ol
1
1
05

0. 1
02

0 . 05
1
ro

ro
0.

0.
0

00

00
00

0.

00
tr
0.

0.
0.
0

0.
0.
0.
0
t

t
on

on

on
C

C
Essential oil (%) Essential oil (%) Essential oil (%)

A. actinomycetemcomitans P. gingivalis H. influenzae

10 4 10 4 10 5

10 3 10 3 10 4
10 3
CFU

CFU

CFU
10 2 10 2
10 2
10 1 10 1 10 1
10 0 10 0 10 0
ol

ol

l
1
1
05
02
0 . 01
0. 5
1

1
0. 1
0. 5
02
0. 1
0. 5

1
1
1

05

0. 2
0. 1
0 . 05
1
ro
00
00

0
00
00

0
0

00
0.

0.

0.
tr

tr
0.
0.
0.

0.

0.
0.

0
nt
on

on

Co
C

Essential oil (%) Essential oil (%) Essential oil (%)

P. aeroginosa
10 4

10 3
CFU

10 2 Figure 27, A.
10 1

10 0
ol
1
1
0. 5
02
0. 0 1
0. 5
1
0.
0

00
00
tr

0.

0.
on
C

Essential oil (%)

45
 S. pyogenes S. aureus L. fermentum
10 5 10 4 10 5
10 4 10 4
10 3
10 3

CFU
CFU
10 3
CFU

10 2
10 2 10 2
10 1 10 1 10 1
10 0 10 0 10 0

1
ol
1

05
02

0. 1
0. 5
1
1
1
05
02

0. 1
0. 5
1
ol
1
1
05
02

0. 1
0. 5
1
ol

0.

0
00
00
0.

0
00
00
0.

0
00
00

tr
tr

0.
0.
0.
tr

0.
0.
0.
0.
0.
0.

on
on
on

C
C
C

Essential oil (%) Essential oil (%) Essential oil (%)

L. acidophilus S. mutans E. faecalis

10 4 10 4 10 4

10 3 10 3 10 3
CFU

CFU
CFU

10 2 10 2 10 2

10 1 10 1 10 1

10 0 10 0 10 0
1
1
05
02

0. 1
0. 5
1
ol

1
1
05
02

0. 1
0. 5
1

1
1
05
02

0. 1
0. 5
1
ol

ol
0.

0
00
00

0.

0.
0
00
00

0
00
00
tr

tr

tr
0.
0.
0.

0.
0.
0.

0.
0.
0.
on

on

on
C

Essential oil (%) Essential oil (%) Essential oil (%)

E. faecium
10 4

10 3
CFU

10 2
Figure 27, B.
10 1

10 0
1
1
05
02

0. 1
0. 5
1
ol

0.

0
00
00
tr

0.
0.
0.
on
C

Essential oil (%)

Figure 27: Dose-response analysis of antibacterial activity of Miswak essential oil.

The numbers of surviving bacteria were determined by counting colony forming units (CFU)
after 90 min co-incubation with essential oil. The essential oil concentration is the percentage of
final assay volume. Control is with DMSO only. The different concentrations correspond to
28Pmole, 2.8 Pmole, 1.4Pmole, 0.56 Pmole, 0.28 Pmole, 0.14Pmole and 0.028 Pmole pure
BITC. A) Gram-negative bacteria, B) Gram-positive bacteria.

46
 I E-Coli MC4100 control II E. coli MC4100 control
E-Coli MC4100 miswak oil 0.02% E. coli MC4100 miswak oil 0.1%
A.a control A.a control
A.a miswak oil 0.02% A.a miswak oil 0.1%
10 4 10 4
10 3
10 3
10 2

CFU
CFU

10 2
10 1
10 1 10 0
10 0 10 - 1
0 2 5 10 20 40 90 0 2 5 10 20 40 90
Time (min)
Time (min)

Figure 28: Assessment of surviving CFU of Gram-negative bacteria E. coli MC 4100 and A.
actinomycetemcomitans HK 1519 in response to two S. persica oil dilutions at the times
indicated. I) Kinetics of bacterial killing with 0.02% oil dilution (= 1.4Pmole pure BITC). II)
Kinetics of bacterial killing with 0.1 % oil dilution (= 2.8Pmole pure BITC). Control was with
DMSO only.

Bactericidal effect of essential oil with increasing ratio bacteria to oil

The results showed that the number of the surviving CFU decreased dramatically
compared to the control with no oil. It also showed that the CFU did not change
proportionally with every ten times increase in bacteria numbers [Table 8].

Table 8: Number of colony forming units (CFU) with increasing number of bacteria and
constant essential oil concentration with 0.1% oil for A. actinomycetemcomitans and 0.05% oil
for E. coli strain MC4100.

Bacteria Time point CFU

(min)
A. actinomycetemcomitans 0 9x104 9x105 9x106 9x107
90 6 4 136 7264
E. coli MC 4100 0 6x105 6x106 6x107 6x108
90 1 4 30 590

47
The pooled fractionation from MPLC
The test of pooled fractions on two Gram negative and two Gram positive bacteria
indicated that the pooled fractions were strong bactericidal against E-coli MC4100 and
A. actinomycetemcomitans, but were not that strong against S. pyogenes, and L.
acidophilus. Fractions 2, 3, and 5 were the most effective against E-coli. Fractions 2
and 3 were the most effective against A. actinomycetemcomitans.

Fractions from 1 to 6 killed 85% of S. pyogenes, while L. acidophilus was not much
affected by the any of the oil fractions. Fractions 7 and 8 had no major impact,
compared to the control. In fact, S. pyogenes growth was enhanced in comparison to the
control. All 8 pooled fractions were remixed together to test for synergistic effect. The
mixed fractions indicated no increased antibacterial capacity on any of the tested
bacteria [Figure 29].

The antibacterial repertoire of the three pooled fractions 2, 3, and 5 was similar to S.
persica essential oil with respect to killing activity against Gram-positive and Gram-
negative bacteria. This emphasised the fact that BITC is the major antibacterial
component of the S. persica root [Figure 30].

Transmission electron microscopy

The electron micrographs of bacteria treated with oil exhibited dramatic effects on the
cell membrane. In the presence of the S. persica essential oil, the bacterial membrane
exhibited protrusions already at 2 minutes incubation time. These protrusions increased
in size and prevalence with increasing incubation time. The commercially available
pure BITC induced similar protrusions on the bacterial membrane. The reference
antibiotic ampicillin, which targets the cell-wall synthesis, did not exert the same cell
membrane protrusion effect on the bacteria [Figure 31].

48
 E-Coli MC4100
A. actinomycetemcomitans
S. pyogenus
Full Fractions data L. acidophilus
12000

10000

8000
CFU

6000

4000

2000

0
ol

d
1

xe
ntr

Mi
Co

Fractions

Figure 29: Antibacterial activity pooled MPLC fractions of S. persica root: Two Gram-
negative (E. coli MC 4100 and A. actinomycetemcomitans) and two Gram-positive bacteria (S.
pyogenes and L. acidophilus) were used for bactericidal activity detection. Controls were with
DMSO only. Pooled fractions 1-8, combined.

E. coli MC4100 A. actinomycetemcomitans

10 4 10 4

10 3 10 3
CFU

CFU

10 2 10 2

10 1 10 1
10 0 10 0
t
ol

ol

ct
ac
tr

a
tr
Fr

Fr
on

on
ed

ed
C

C
ix

ix
M

Pooled Fraction no. Pooled Fraction no.

L. acidophilus
10 5 S. pyogenus 10 4
10 4 10 3
CFU
CFU

10 3
10 2
10 2
10 1 10 1
10 0 10 0
l

t
2

5
ol

tro

rac
r ac
n tr

dF
dF

Co
Co

xe
xe
Mi

Mi

Pooled Fraction no. Pooled Fraction no.

Figure 30: Active pooled fractions of S. persica root. Controls were with DMSO only. Pooled
fractions 1-8, combined.

49
Figure 31: Transmission electron micrographs of A. actinomycetemcomitans treated with S.
persica essential oil, synthetic BITC or Ampicillin. Samples were withdrawn at time points
indicated. Concentration of essential oil was 0.1% corresponding to 2.8 μmole BITC, synthetic
BITC was 2.8 μmole and ampicillin was 1 mg/ml. Control for essential oil and BITC was with
1% DMSO. For ampicillin, the control was with 1% water.

50
DISCUSSION
Our findings supported the hypothesis that Miswak stick pieces, without extraction,
have a strong antibacterial effect against most bacterial species tested. Corresponding
or greater antibacterial effects were associated with Miswak suspended 3 mm above the
inoculated agar plates, suggesting the presence of volatile active antibacterial
compounds.

The inhibition zones associated with the Miswak pieces clearly demonstrated much
stronger inhibitory effects than the aqueous Miswak extract. For example, for S.
mutans, the Miswak pieces caused unexpectedly large inhibition zones of 3.4 cm (2.6-
4.6); whereas, our preliminary tests of aqueous Miswak extract yielded an inhibition
zone against S. mutans of only 0.2 cm. This result was in accordance with earlier
studies in which 50% Miswak aqueous extract achieved an inhibition zone of 0.2-0.3
cm (Al-Lafi and Ababneh, 1995; Almas et al., 1997; Almas, 1999; Almas and Al-
Bagieh, 1999). The weak antibacterial effect observed with aqueous Miswak extract
suggested that the active compounds were not extracted or were deactivated during
preparation of the crude aqueous extract. Using this kind of extract may probably not
reflect the real antibacterial activity of Miswak.

The antibacterial effect of Miswak pieces on A. actinomycetemcomitans, P. gingivalis,

H. influenzae and L. acidophilus cannot be compared with the crude extract, as there
are no published studies on the effect of Miswak extract on these bacterial strains. It
was also difficult to compare the antibacterial effect of Miswak pieces with that of
known antibacterial substances as the exact content and amount of Miswak pieces is
unknown. Thus, the need to evaluate Miswak antibacterial effect by standard methods
of evaluating antibacterial substances prompted an alternative extraction method for
obtaining an active Miswak extract.

Pharmacological studies indicated that with steam distillation it is possible to obtain

essential volatile oil from the roots, stems, and leaves of Salvadora Persica (Bader et
al., 2002; Alali and Al-Lafi, 2003; Alali et al., 2004). However, no studies were
conducted to test the antibacterial activity of the root oil on oral bacteria. From GC-MS
analyses, the root oil comprises mainly of Benzyl isothiocyanate (BIT) (70%),
limonene (9.4%), Į-pinene (8.7%) and flavonoids (2.55%) (Abdel-Wahab et al., 1990;
Bader et al., 2002). Some of these compounds are known to have antibacterial activity.
Studies on BITC and flavonoids separately proved that they have antibacterial,
antifungal, and antiviral activities (Al-Bagieh and Weinberg, 1988; Abdel-Wahab et al.,
1990; Al-Bagieh, 1992; Al-Bagieh, 1998; Cushnie and Lamb, 2005).

The Miswak exhibited stronger antibacterial activity against Gram negative bacteria
than Gram positive bacteria, as evidenced by the pronounced differences in inhibition
zones associated with the Gram negative species A. actinomycetemcomitans, P.
gingivalis, H. influenzae and the Gram positive species S. mutans and L. acidophilus.
Studies on the effects of BITC and flavonoids on Gram negative and Gram positive

51
bacteria present contradictory results (Cushnie and Lamb, 2005). This may be due to
the different assays used to test antibacterial effects and to variations within each assay.
Well standardised studies are needed to identify which components of the oil extract
have an antibacterial effect against Gram negative and Gram positive species.

Comparison of the effect of suspended and embedded Miswak pieces disclosed that the
suspended Miswak pieces had similar or stronger effects on Gram negative bacteria,
whereas, the opposite was true for Gram positive bacteria where the effect of the
suspended Miswak 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 Miswak compounds and their reactions
with surrounding environments is important in order to determine the best method
available for testing its antibacterial activity. The effect of suspended 0.14 g Miswak on
P. gingivalis was so strong that all of the 14 cm agar plates had complete growth
inhibition. However, our laboratory facilities did not allow anaerobic incubation of
larger agar plates. In order to verify that the strong inhibitory effect of 0.14 g pieces
was a real effect of the Miswak and not an artefact, a dose response experiment was
conducted. This experiment revealed a linear correlation between Miswak weight and
inhibitory effect (R2 = 0.99).

H. influenzae is a well characterised upper respiratory tract commensal in humans and

associated animals. It is a major etiological agent of upper respiratory tract infections
and acute exacerbations of chronic bronchitis (Samaranayake, 2006). Initially, H.
influenzae served as a control for the incubation environment of A.
actinomycetemcomitans. However, when the Miswak presented a strong antibacterial
effect against H. influenzae, it was necessary to include these results in the study.

The diverging reports on the chemical nature and antimicrobial capacity of Miswak
sticks prepared from the roots of S. persica, and the strong and air borne antimicrobial
activity achieved in this work, prompted systematic chemical characterisation and
antibacterial testing to identify the antimicrobial components present and investigate
their properties for possible use as future therapeutic antimicrobials.

The volatiles from cut Miswak chewing-sticks from the root of S. persica are highly
antimicrobial, these volatiles consisted of >98% (determined by GC-MS) of benzyl
isothiocyanate (BITC). The steam-distilled essential oil generated from S. persica roots
contained 78% BITC and this essential oil exhibited high antimicrobial activity, mainly
against Gram-negative proteobacteria. With MPLC, the contents of S persica milled
roots were fractionated. The fractions that exhibited the highest antimicrobial activity
had BITC as a dominating component and displayed the same repertoire of bacterial
killing as with the oil. Through three different methods of preparation, BITC was
identified as the main antimicrobial factor in the root of the shrub S. persica. Although
there are naturally other, more polar, components in the roots that could be obtained
through water or alcohol extraction, and which may contribute to the bactericidal
activity, these components are far less potent in antimicrobial capacity and have low
activity against Gram-negative bacteria (Al-Bagieh et al., 1994; Darout et al., 2000b;
Almas et al., 2005; Hammad and Sallal, 2005; Darmani et al., 2006).

52
Upon plant tissue damage, BITC is released as an effector molecule of the plant
defence system through hydrolysis of benzyl-glucosinolate by the enzyme myrosinase,
(Rask et al., 2000). The plant enzyme myrosinase and its substrates glucosinolates are
physically separated in plants, and therefore, plant tissues damage is a prerequisite for
releasing isothiocyanates (Al-Bagieh et al., 1994; Holst and Williamson, 2004):
Cruciferous vegetables of the genera Brassica and Sinapis are rich in benzyl-
glucosinolates. Hence, BITC is released into the oral cavity and gastrointestinal tract
upon consumption of uncooked cruciferous vegetables. The same mechanism appears
active in the release of BITC when a Miswak stick is chewed on prior to mechanical
cleansing of the teeth. As boiled Miswak sticks lose antibacterial activity, most likely
due to inactivation of the myrosinase, this assumption is confirmed.

It has previously been shown that BITC exerts antimicrobial activity; however, the
reported repertoire of susceptible bacteria and killing efficiency differs, probably due to
variations in the methods used for antimicrobial testing (Aires et al., 2009; Beevi et al.,
2009). Earlier, zone-inhibition assay was used, but the medium-containing agar
appeared to inhibit the diffusion of the substances released from pieces of Miswak
sticks. To avoid this inhibitory effect the essential oil or dilutions thereof to bacterial
suspensions were used and BITC-containing essential oil exhibited dose-dependant
antimicrobial activity against a number of medically important Gram-negative bacteria.
The Gram-positive bacteria, all belonging to the firmicutes phyla, were more resistant
or totally unaffected by the oil. Of special interest were the Gram-negative oral
pathogens A. actinomycetemcomitans and P. gingivalis, which exhibited high
sensitivity to the essential oil. This suggested the use of Miswak chewing-sticks for
cleaning teeth may be protective against oral pathogens strongly associated with the
pathogenesis of periodontal disease and tooth loss.

The kinetics of bacterial killing mediated by the essential oil was rapid. Within minutes
the bacteria load was diminished by 1000 times. The short exposure required for
bactericidal effect supported the assumption Miswak chewing-sticks may be efficient
for improving oral health. There are few studies evaluating the in vivo effect of the
practice of Miswak sticks on oral health and conclusions vary with study design
(Darout et al., 2000a; Al-Otaibi et al., 2004). Larger studies with DNA-based analysis
of the microbiota would be necessary in order to evaluate the benefit of Miswak
chewing sticks in oral hygiene.

The rapid killing mediated by S. persica root essential oil suggested BITC-containing
oil might target the bacterial membrane. Electron micrographs of the Gram-negative
bacteria A. actinomycetemcomitans displayed protrusions of the bacteria cell
membrane. Similar membrane protrusions were found with synthetic BITC at the same
concentration as in the essential oil. This membrane effect resembled those reported for
antimicrobial peptide treated bacteria (Lehrer et al., 1989) and the rapid antimicrobial
effect (Boman, 2003). Antimicrobial peptides bind to and destabilise bacterial
membranes and during this process the peptides are essentially consumed (Shai, 2002).
This mechanism of killing confers a stochiometric relation between required numbers
of bound peptide molecules in order to kill a certain number of bacteria (Steiner et al.,
1988). Our results on BITC bactericidal effects with increasing number of bacteria

53
suggested the essential oil targeted a vital enzymatic process rather than exerting its
effect through binding to the bacterial membrane.

It has been demonstrated that the electrophilic end group of BITC can interact directly
with glutathion-reductase and thioredoxin-reductase enzymes that are essential for
maintaining the redox-potential in the cell (Hu et al., 2007). Interestingly, in a recent
publication Daniels et al (2010) suggest Gram-positive bacteria of the firmicutes phyla
exclude cysteins in their secreted and cytoplasmic proteins at a gene level, as a means
of coping with a highly oxidative environment. This was in contrast to Gram-negative
proteobacteria, in which cystein-containing secreted proteins mature in the oxidative
periplasm assisted by glutathion-like oxidoreductases for disulfide bond formation
(Nakamoto and Bardwell, 2004). One possible mechanism for BITC bacterial killing
could be that BITC, which is rather lipophilic but also has electrophilic properties, may
pass the cell membrane and bind to the bacterial glutation-like oxidoreductases, such as
the Dsb family (Nakamoto and Bardwell, 2004), thus, interfering with the bacteria cell
redox-potential. The repertoire of bacteria killed by BITC, Gram-negative
proteobacteria and the anaerobic Gram-negative P gingivalis of the bacteriodetes phyla,
would concur with this speculation

Naturally occurring isothiocyanates released during consumption of cruciferous

vegetables have demonstrated cancer-preventive activity in animal models, and in
epidemiological studies, high dietary intake of isothiocyanates is associated with a
reduced risk for lung, colorectal, breast, and prostate cancer in humans (Fenwick et al.,
1983; Conaway et al., 2002; Wu et al., 2004). There are numerous reports on the
chemoprotective activity of synthetic BITC (Miyoshi et al., 2004; Zhang and Gordon,
2004; Warin et al., 2009) and some also address the possible adverse effects of BITC
(Kassie et al., 1999). Interestingly, genotoxic effects of BITC in terms of DNA damage
are demonstrated in vitro on liver- and gastric cell lines at low doses (5Pg/ml) after one
hour of exposure. This is contrary to the in vivo results in which doses of 220 mg
BITC/kg body weight are required to cause a shift in the DNA migration pattern of rat
gastric and colonic mucosa cells. The discrepancy between in vivo and in vitro data
suggests that BITC is detoxified in the living organism (Munday and Munday, 2004).
Indeed it has been demonstrated that the chemopreventive activity of BITC is partially
through activation of detoxification systems (Shapiro et al., 1998). Together these data
concerning the effects of BITC on eukaryotic organisms suggests the possible
antimicrobial therapeutic interval of BITC is viable for further investigation due to the
rather high in vivo tolerance and the documented positive health effects when BITC is
ingested via the consumption of vegetables.

54
MAIN FINDINGS
x Rinsing with Miswak extract resulted in protracted elevation of plaque pH (>6.0).
The difference in plaque pH between Miswak extract and water rinse was
statistically significant at 30 minutes (p< 0.01).
x Miswak water extract stimulated parotid saliva flow rate significantly (p< 0.01).
x Toothbrushing with fresh Miswak significantly reduced dental plaque and gingival
inflammation while the deactivated Miswak did not.
x Both fresh and deactivated Miswak reduced approximal plaque significantly, but
there was no significant change in the approximal gingival index.
x Testing the antibacterial effect of fresh Miswak pieces embedded in inoculated agar
plates indicated Miswak had very strong antibacterial activity.
x The inhibitory effect was most pronounced on P. gingivalis, A.
actinomycetemcomitans, and H. influenzae, less on Streptococcus mutans and least
on Lactobacillus acidophilus.
x Suspending Miswak pieces on top of inoculated agar plates had a comparable effect
to the embedded Miswak pieces.
x A dose response effect was demonstrated when different Miswak weights were
tested on P. gingivalis.
x Miswak essential oil had a strong antibacterial activity against seven Gram negative
bacterial strains.
x Benzyl isothiocyanate is the main antimicrobial component of Miswak from
Salvadora persica roots. Synthetic Benzyl isothiocyanate had comparable
antibacterial activity to Miswak essential oil.
x Electron microscopy revealed bacterial cell membrane exhibited protrusions upon
treatment with Miswak essential oil and synthetic Benzyl isothiocyanate. The
bacterial cell membrane presented different changes when treated with ampicillin.

55
CONCLUSIONS
Miswak extract from Salvadora persica raised plaque pH after acidic challenge; this
elevation was maintained until the end of the testing session. Previous studies report an
immediate reduction of S. mutans count and a significant increase in calcium and
chloride concentrations. These findings together with the protracted elevation of plaque
pH determined in this thesis suggested Miswak might have a positive roll in the
reduction and prevention of dental caries. Fresh Miswak had a significant effect on
dental plaque and gingival inflammation, suggesting that Miswak might play a
potential role in periodontal disease prevention. However, fresh Miswak did not have
superior properties to deactivated Miswak on interproximal embrasures, which are
difficult to access, suggesting a limited chemical effect on the microflora in our study
population. Therefore, Miswak can be used as a dental hygiene method for maintaining
good oral hygiene; however, additional interproximal cleaning aides are needed in
combination with Miswak use.

Miswak prepared from the roots of Salvadora persica had strong antibacterial activity
when tested without extraction. The experiment with pieces of Miswak suspended over
the agar plate revealed the antibacterial component was volatile. Miswak oil, extracted
with steam distillation, had the same strong antibacterial effect as the root per se. Gas
chromatography and fractioning identified Benzyl isothiocyanate as a major
antibacterial effector molecule of Salvadora persica root. Benzyl isothiocyanate is
released from the root upon tissue damage and when Miswak sticks made from
Salvadora persica root are used for oral hygiene, an antibacterial effect is obtained
fresh and instantaneously. The difference in bacterial cell wall change when treated
with Benzyl isothiocyanate and ampicillin suggested Benzyl isothiocyanate killing
mechanism of action is different to that of ampicillin.

FUTURE PERSPECTIVES
Further studies are warranted for exploring and identifying the underlying mechanisms
of action of Miswak on bacteria. The strong and rapid antimicrobial effect against
Gram-negative gammaproteobacteria, a class of bacteria that include medically
important pathogens such as Salmonella enterica, Porphyromonas gingivalis and
Haemophilus influenzae, advocates in favour of investigating the potential of Benzyl
isothiocyanate as an antimicrobial substance for therapeutic use. The volatile character
of Benzyl isothiocyanate might be an avenue for exploring applications where confined
body spaces require antibacterial treatment. Studying the effect of brushing with
Miswak on populations with periodontitis, having a predominantly Gram-negative
flora, would be necessary for testing the effectiveness of Miswak in the treatment and
prevention of periodontal disease. To test a chemical antibacterial effect of Miswak
without mechanical interference would require the use of an extract rather than the
sticks per se. Laboratory and clinical investigations of antiviral and antifungal activities
together with the effect on periodontal bacteria need to be performed. A possible anti-
inflammatory effect of a Miswak extract should also be explored. The cytotoxic

56
activities of fresh Miswak and Miswak oil need to be evaluated before the development
of oral applications becomes a future reality.

57
ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to everyone who has supported and
encouraged me and made it possible for me to complete this thesis. My warm thanks
to all of you. I would like to especially thank:

I am heartily thankful to Professor Anders Gustafsson, my principal supervisor, his

encouragement, guidance, and support from the initial to the final level enabled me to
develop an understanding of the subject and achieve my goal. Thank you Anders, I
have learned from you not only how to conduct scientific research but also how to
manage my life through difficult times. Thank you for being so kind.

My great thanks to my co-supervisor Professor Peter Lingström, for his unique,

distinguished help, and always being there for support and discussions. You are a
supervisor that every student dreams of.

I would like to thank Professor Ann-Karin M Borg Karlsson, for her broad
knowledge and constructive comments. You have provided the basis for achieving the
good results I got in the chemistry part of my project.

Professor Peter Baeckström, thank you for your sincerity, guidance, and remarkable
influence on my work. I really enjoyed all the discussions we had, the scientific and
non-scientific ones, you made really hard times enjoyable.

I also wish to thank my colleagues and friends at the Department of Organic

Chemistry, Royal Institute of Technology: I thank Dr. Katinka Pålsson for her gifted
and talented work in the preparation of the miswak essential oil, Ellen, for her
unlimited help and support, Azeem, for always being there for all my questions and
work in chemical analysis.

I owe my most sincere gratitude to Dr Katrin Pütsep, who gave me the chance to
work in her microbiology lab. Katrin gave me untried help during my difficult
moments. Her kind friendly support always pushed me forwards to accomplish this
thesis. Thank you Katrin, without your presence beside me all the time this work
could not be finished.

My special gratitude to my best friend Dr Fernanda Brito, who gave me scientific

support, help, and love and came from Brazil to help in my thesis. Fernanda, thank
you for being so close, when you were very far. I am deeply indebted to my best
friend Dr Nadia Al-Qadi, for her great role and support and being beside me and
maintaining communication from the United Arab Emirates.

My gratitude is extended to Jane Harrison-Wahlen, from Germany for her

competent technical help in transporting my samples from Saudi Arabia to Sweden.
Without her sincere help, it would have been impossible to complete my fourth paper.
I would like to thank Dr Fadi Al hemsi, for his technical guidance and support.

58
I would like deeply to thank Sue Pajuluoma, for her precise, neat, and fast revision
of the English of my thesis. Thank you Sue for being so kind.

Through every stage I have my full stream from my father Professor Hamed Sofrata,
Dad, you helped me a lot in statistical analysis, formatting my thesis, in every single
step. There are no words that adequately express my appreciation to you. Thank you
for having faith in me.

I thank my children Esra, Yosra, and Mohammad, for being patient, for sharing me
with my work, and for bringing a smile to my heart. To my family: thank you all for
giving me the courage and strength to pursue my dreams. I love you very much.

Finally, to my husband Mahmoud, I am indebted to you for editing numerous drafts

with a mighty pen and for the countless conversations and debates about my research,
resulting in a better organised and better argued book. In many ways, this has been a
joint achievement. Most of all, thank you for being there for me all these years,
encouraging me to forge ahead while finding a balance in my life by being that balance
in my life.

59
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