26

In vitro antimicrobial effects of crude miswak extracts on oral pathogens

Howaida F. AbdElRahman*, BSc, M Phil Nils Skaug‡, DDS, PhD
†
George W. Francis , Dr ing, Fil dr

In vitro, epidemiological and clinical studies have demonstrated beneficial effects of chewing sticks on oral hygiene. The aim of this
study was to assess under standardized test conditions whether miswak crude extracts prepared from S. persica roots and twigs
using different solvents inhibited in vitro growth of some selected oral microbes involved in infections in humans. Sterile distilled
water, 96% ethanol, 2% acetic acid and ethyl acetate were used as solvents. Reference strains of Streptococcus mutans,
Lactobacillus acidophilus, Actinobacillus actinomycetemcomitans, Actinomyces naeslundii, Porphyromonas
gingivalis, Prevotella intermedia, and Candida albicans were tested for susceptibility to the antimicrobial effects of crude
extracts using the broth microdilution method of Cai et al. Microbial growth was estimated spectrophotometrically at 650 nm in 96-
well microtiter plates. Different concentrations of the S. persica extracts were incubated at 37oC with each test strain for up to 72 hrs.
The minimum inhibitory concentration (MIC) of the extracts against the individual test organisms was determined as the lowest
concentration of the extract that limited turbidity to < 0.05 absorbance at OD650nm. Results showed that the root-ethanolic extract was
the most potent. The most susceptible strain was S. mutans whereas L. acidophilus was the least susceptible. MIC values for the
various plant extracts ranged from 100mg/ml to 300 mg/ml. Based on these results, it was concluded that miswak extracts exhibited
low antimicrobial activity against the test microorganisms when compared with 0.2% aqueous chlorhexidine.

Introduction (Sinkat), Kassala (Gedaref and Dinder), White Nile

(Dueim, Getaina), Khartoum and Kordofan and its
main usage is currently as miswak for oral
The World Health Organisation has cleansing.2,3 The promotion of good oral health by
recommended and encouraged the use of miswak is mainly attributed to mechanical
chewing sticks.1 Recently, chewing sticks have cleansing, but may also be due in part to built-in
been comprehensively reviewed 2 , 3 and antiseptics.7
examination of their effectiveness as an oral "Siwak purifies the mouth and pleases Allah”
hygiene aid has been encouraged.4 The Arabic Prophet Muhammad (PBUH) said.8 Islam teaches
word miswak is used to describe a chewing stick the importance of cleanliness of the body as well
used for cleaning teeth, tongue and gum. Miswak as the mind and therefore introduced basic oral
includes all types of sticks used as oral cleansing hygiene by incorporating it as a religious practice.9
aids. In Sudan, miswak is prepared from stems, The religious and spiritual impact of miswak
roots or twigs of Salvadora persica L. (order: probably is the principal reason why it is
Celastrales, family: Salvadoraceae). This shrub is extensively used in Islamic countries.
commonly known as the arak tree5 and has a wide Different parts of the plant have shown various
geographical distribution over the Middle East and chemical components when analyzed by different
most of the African countries.6 In Sudan, it is methods.10-14 Several in vitro studies have
distributed in the arid areas of the flood plains indicated that S. persica contains substances that
along valleys and seasonal water courses known possess dental plaque-inhibiting and anti-
as “khors” in North and East Sudan, Red Sea Hills microbial properties against oral microbes.15-21
Received 30 April 2001; Revised 6 June 2001 The aim of this study was to compare the
Accepted 23 July 2001 antimicrobial effects of S. persica root and twig
*Former M. Phil. student, Centre for International Health extracts using different solvents, standardized test
University of Bergen
‡
Professor of Oral Microbiology, Institute of Odontology Oral Address reprint requests to:
Microbiology, University of Bergen Dr. Howaida Faisal AbdElRahman
†
Professor of Organic Chemistry, Faculty of Natural Sciences P.O. Box (1113) 6054
and Mathematics Department of Chemistry, University of Khartoum, Sudan
Bergen, Bergen, Norway E-mail: [email protected]

Saudi Dental Journal, Vol. 14, No. 1, January - April 2002

ABDELRAHMAN ET AL. 27

conditions and a panel of target microorganisms mg/ml and 200 mg/ml respectively. They were
known to be involved in oral and systemic then centrifuged at 15800 xg for 20 min using a
infections in humans. cooled centrifuge** at 10oC. The supernatants
were sterilised by filtration through a 0.2 mm
Materials and Methods membrane filter‡. Each stock solution was used to
prepare two 2-fold serial dilutions. The pH of the
S. persica authenticity, collection and grinding reconstituted extracts were determined using a
pH-meter++.
Roots and twigs of S. persica were collected
from Khor Adeit, Sinkat (north-eastern Sudan) in Cultivation of microbes
July 1999. Two plant taxonomists from the Depart-
ment of Botany, University of Khartoum and School The following type-strains were used for the
of Life Sciences, University of ElNeelen, Sudan, antimicrobial testing of the crude extracts:
recommended this location since the plant is the Actinobacillus actinomycetemcomitans ATCC
dominant species in the area.6 Prior to collection, a (American Type Culture Collection) 43717,
visit was made to the Botanical Garden of Actinomyces naeslundii 40110/87, Candida
Khartoum to study the cultivated plant and the wild a l b i c a n s ATC C 9 0 0 2 8 , L a c t o b a c i l l u s
types in the area of collection. An agriculturist and acidophilus CCUG (Culture collection University
some inhabitants of the area helped to identify of Gothenburg, Sweden) 5917, Porphyromonas
these. After collection, the plant material was air- gingivalis W50 Black, Prevotella intermedia
dried in a shaded area and then air-shipped to the VPI (Virginia Polytechnical Institute) 4197 and
Laboratory of Oral Microbiology, University of Streptococcus mutans CCUG 11877. Fresh
Bergen, Bergen, Norway, where the antimicrobial cultures were prepared from the strains stock
examinations were performed. Each plant sample cultures. All the bacterial strains were grown on
was cut into small pieces using plant scissors and blood agar (1.2% agar with 5% sheep blood) for
then finely ground using a grinder*. The powders anaerobes (LAB M fastidious anaerobes agar) and
were kept separately in sterile, dry screw-capped aerobes (Columbia agar Difico 0790-17), while the
bottles which were stored in a dry and cool place, yeast was cultivated on Sabouraud dextrose agar
until extraction. (Oxoid CM 41). P. gingivalis, P. intermedia, L.
acidophilus and A. naeslundii were incubated
Extraction and reconstitution anaerobically while S. mutans and A.
actinomycetemcomitans were incubated
Sterile distilled water, 96% ethanol, ethyl microaerophilically for 48 hrs using the Anoxomat
acetate and 2% acetic acid were used for crude
System‡‡ and C. albicans was incubated
extractions. The extracts were prepared by mixing
aerobically at 370C for 24 hrs.
50 g plant powder and 250 ml solvent in sterile, dry
screw-capped bottles. 18 The bottles were
Antimicrobial susceptibility testing
maintained at room temperature in a shaker+ at
400 rotations per min. The solvents were changed
every 24 hrs for 9 days, and the supernatants were The microdilution method of Cai et al.22 was
separately collected in sterile screw-capped applied using 96-well microtiter trays***. The
bottles and kept at 4-600C. The volume of each growth medium employed was tryptic soy broth
extract was reduced by evaporation under (3%)-yeast extract (0.5%) media (TSB-YEM) (Difico
pressure at 35-380C and the remaining solvent was 0370-17-3) supplemented with cys teine
removed by air-drying for 2-4 days at room hydrochloride (0.05%), menadione (0.02μg/ml),
temperature. The yield of each extract was hemin (5μg/ml) and 0.02% potassium nitrate
calculated as a percentage of the original weight. (supplemented TSB-YEM). Innocula were prepared
The dried extracts were then kept in a dry place at for all test microbes to obtain about 3 x 105 colony
40C (in a cold room) until its use for antimicrobial forming units of test microbes in the growth
testing. Before testing, each crude extract was medium. Suspension in sterile saline of each
freshly reconstituted in 0.5% Tween 80Ò to prepare **Sorvall centrifuges, Wilmington, Delaware, USA
‡Schleicher & Schüell, Germany
two stock solutions with concentrations of 300 ++Schott, type CG842, Germany
‡‡MARTÒ Microbiology Automation, The Netherlands
*Mikro-Feinmühle-Culatti, Janke & Kunkel GMBH & Co. KG, IKAÒ
- labortechnik, Germany ***NUNCTM Brand products, Nalge Nunc International,
+Edmund Bühler 7400 Tübingen, Germany Denmark

Saudi Dental Journal, Vol. 14, No. 1, January - April 2002

28 CRUDE MISWAK ON ORAL PATHOGENS

microbial test strain was adjusted to McFarland persica extract (mg/ml) or control (v/v %) added
standard 1 (»3x108 bacterial cells). A volume ratio to each well.
of strain suspension in saline to medium (1μl:1ml) Spot samples taken from microtiter plate wells
was prepared according to the amount needed per with visible growth were cultivated on blood agar
analysis. In different wells, 100μl of each for the bacteria and on Sabouraud dextrose agar
innoculated or uninnoculated (background wells) for C. albicans to check for microbial contami-
TSB-YEM was added to 100μl of each of the crude nation. Gram-stained smears of the samples were
extract concentrations or controls. Serial dilutions prepared and examined microscopically using oil
of chlorhexidine in sterile distilled water (final immersion at 1000 x magnification.
concentration 0.005%-0.0001562%) and tea tree oil
in 0.5% Tween 80Ò (final concentration 0.5%- Results
0.0625%) served as positive controls. Negative
controls were sterile saline, medium without Antimicrobial susceptibility testing
additives and 0.5% Tween 80Ò. Each test was
carried out in triplicate. Innoculation times were The extraction yields ranged from 38.8 % for
determined from growth curves obtained the root-acetic acid extract to 3.3% for the stem-
individually for each microorganism in pilot ethyl acetate extract. The pH range was from 5.9
experiments. According to this, the trays were for the stem-water extract to 3.9 for the root-
incubated at 370C for up to 72 hrs; C. albicans ethanolic extract. The pH of chlorhexidine
aerobically, S. mutans and A. actinomy- standard solutions was 6.6.
cetemcomitans were incubated microaero- The crude extracts either inhibited or enhanced
phillically, and the remaining strains were the growth depending on which test strain was
incubated anaerobically. Growth was estimated used (Table 1). There was no growth for P.
spectrophotometerically as turbidity by measuring intermedia in the final medium. S. mutans was
the light absorption of the microbial mass as the most susceptible strain to all extracts while L.
determined by the optical density (OD) readings at acidophilus was resistant to all extracts except for
650 nm using a microtiter plate reader**. Growth the root-ethanolic extract. Compared with the
was checked at 0 hrs, 24 hrs and 48 hrs, and at 72 other solvents, the ethanolic extracts showed the
hrs for the slow growing strains P. gingivalis and strongest antimicrobial activity. Within the
P. intermedia. ethanolic extracts the root extract was more potent
Values calculated from the differences in than the twig extract. The stem-water extract was
OD650nm readings between inoculated TSB-YEM found to have the least effect. Saline and Tween
(the medium plus crude extract or control and 80Ò (serving as negative controls) showed
bacteria) and uninoculated TSB-YEM (the medium negligible reduction in the OD650nm readings. As
plus crude extract or control alone) wells were examples of graphically presented effects of crude
used to assess the susceptibility of each strain to extracts on the test bacteria after 48 hrs
the different crude extracts. The antimicrobial incubation, Figs. 1 and 2 show different effects of
activities were presented graphically as the mean root and stem extracts, respectively, on S. mutans
of the triplicate OD650nm differences between and Figs. 3 and 4 on L. acidophilus.
wells with growth and background wells, y-axis; The MIC values of the various crude extracts
the x-axis showing the medium alone or with the and the positive controls varied with the test
different additives. The growth inhibition was microbes. The MIC values ranged from 100 mg/ml
assessed by subtracting OD650nm values of to 300 mg/ml for the different crude extracts (Table
incubated medium supplemented with S. persica 2). MIC values were not determined where no
extracts or controls from those of incubated inhibition of growth was observed.
medium alone. The minimal inhibitor y
concentration (MIC) of each extract for each strain Discussion
tested was defined as the lowest concentration of
the extract that limited the turbidity to <0.05 According to the findings of this study, the S.
absorbance at 650 nm.22 These values were persica crude extracts inhibited, reduced or
calculated according to the volume (100μl) of S. enhanced the growth of the test microorganisms.
Most of the extracts exerted their antimicrobial
**Molecular DeviceÒ/E max/ for MacintoshÒ / version 2.3, activity only at the highest concentrations used
Molecular Devices Corporation, Sunnyvale, CA, USA while chlorhexidine and tea tree oil showed

Saudi Dental Journal, Vol. 14, No. 1, January - April 2002

ABDELRAHMAN ET AL. 29

Table 1. Antimicrobial activity of different S. persica extracts.

Stem extracts

Microorganisms Incubation Ethanol Water Ethyl acetate Acetic acid

periods (96%) (2%)

C. albicans 24 Reduction Reduction Reduction Inhibition

48 Reduction Reduction only at the Enhancement Inhibition
highest concentration
otherwise no effect
72 Not carried out Not carried out Not carried out Not carried out

S. mutans 24 Enhancement Enhancement Inhibition Inhibition

48 Inhibition Reduction Inhibition Inhibition
72 Not carried out Not carried out Not carried out Not carried out

A. actinomycetem- 24 Inhibition Enhancement Inhibition Inhibition

comitans 48 Inhibition Enhancement Inhibition Inhibition
72 Not carried out Not carried out Not carried out Not carried out

L. acidophilus 24 Enhancement Enhancement Enhancement Enhancement

48 Enhancement Enhancement Enhancement Enhancement
72 Not carried out Not carried out Not carried out Not carried out

A. naeslundi 24 Inhibition Enhancement Inhibition Inhibition

48 Inhibition Reduction Inhibition Inhibition
72 Not carried out Not carried out Not carried out Not carried out

P. gingivalis 24 Not carried out Not carried out Not carried out Not carried
out
48 Inhibition Inhibition Not carried out Reduction
72 Inhibition Inhibition Not carried out Reduction

Root extracts

Microorganisms Incubation Ethanol Water Ethyl acetate Acetic acid

periods (96%) (2%)

C. albicans 24 Inhibition Enhancement Enhancement Enhancement

48 Inhibition Reduction in the highest Enhancement Enhancement
concentration otherwise
no effect
72 Not carried out Not carried out Not carried out Not carried out

S. mutans 24 Inhibition No growth was indicated Inhibition Inhibition

48 Inhibition Inhibition Inhibition Inhibition
72 Not carried out Not carried out Not carried out Not carried out

A. actinomycetem- 24 Inhibition Inhibition Inhibition Inhibition

comitans 48 Inhibition Inhibition Inhibition Inhibition
72 Not carried out Not carried out Not carried out Not carried out

L. acidophilus 24 Inhibition Enhancement Enhancement Enhancement

48 Inhibition Enhancement Enhancement Enhancement
72 Not carried out Not carried out Not carried out Not carried out

A. naeslundii 24 Inhibition Inhibition Inhibition Inhibition

48 Inhibition Inhibition Inhibition Inhibition
72 Not carried out Not carried out Not carried out Not carried out

P. gingivalis 24 Not carried out Not carried out Not carried out Not carried
out
48 Inhibition Inhibition No growth was Enhancement
indicated
72 Inhibition Inhibition No growth was Enhancement

Saudi Dental Journal, Vol. 14, No. 1, January - April 2002

30 CRUDE MISWAK ON ORAL PATHOGENS

Table 2. Minimal inhibitory concentration* of different S. persica crude extracts against various test microorganisms as determined
by the Cai (1996) method.
Extracts
Strains Ethanol (96%) Water Acetic acid (2%) Ethyl acetate
Root Stem Root Stem Root Stem Root Stem

C. albicans 150

A. naeslundii 100 300 100 100 300

L. acidophilus 200

S. mutans 50 150 300 200 300 200

A. actinomycete-
mcomitans 50 100

P. gingivalis 150 7 7

P. intermedia 7 7 7 7 7 7 7 7
Key of the table: 7 = no growth on the final medium, = no minimal inhibitory concentration (MIC) was established,
*The MIC of each extract for each strain tested was defined as the lowest concentration of the extract that limited the turbidity to

Medium and additive Medium and additive

Fig. 1. Effect of S. persica root extracts on the growth of S. Fig. 3. Effects of S. persica root extracts on the growth of L.
mutans after 48 hrs microaerophilic incubation at 370C.* acidophilus after 48 hrs aerobical incubation at 370C.*

Medium and additive Medium and additive

Fig. 2. Effect of S. persica stem extracts on the growth of S. Fig. 4. Effects of S. persica stem extracts on the growth of L.
mutans after 48 hrs. microaerophilic incubation at 370C. * acidophilus after 48 hrs. aerobic incubation at 370C.*
*The curves indicate experiments performed on four different days. Medium=medium (TSF-YEM) alone; R/Tw80 and S/Tw80=root (R)
extract and (S) extract, respectively, reconstituted in 0.5% Tween 80Ò; CDX=0.2% chlorhexidine; and TTOil/Tw80=tea tree oil diluted in
0.5% Tween80Ò.

Saudi Dental Journal, Vol. 14, No. 1, January - April 2002

ABDELRAHMAN ET AL. 31

inhibition and growth reduction, respectively, at 420 nm, respectively, at macroscale. Other studies
much lower concentrations. S. mutans was the utilised the agar diffusion method which was also
most susceptible strain to all the extracts and L. tried unsuccessfully in our laboratory.25 A
acidophilus was the most resistant. Regarding problem, however, arises when comparing our
inhibition, the most potent extract was the root- results with those of the studies which used agar
ethanolic extract and the weakest one was the diffusion tests. The latter did not quantify the
stem-water extract. Their effects on growth were microbial inhibition neither did they report MIC
most likely due to the release of chemicals from values. Only one of the studies used a standard
the crude extracts into the medium when they a n t i m i c ro b i a l a g e n t fo r c o m p a r i s o n . 19
were mixed. The different reactions of each strain Consequently, the effective antimicrobial activity
to the various extracts indicated that each solvent reported by the authors who used the agar
extracted different chemical components of S. dif fusion tes t needs verification using
persica (miswak). The strength of the standardised testing and other methods. Our
antimicrobial activity may also depend on the pH study was such an attempt.
of the extracts since the lowest pH was shown by
the root ethanolic extract while the stem water Conclusion
extract demonstrated the highest pH. This
assumption is in agreement with a recent study by Based on our results, we concluded that:
Almas in 1999.15 However, according to our
findings, it appears that miswak has a relatively 1. Crude miswak extracts showed low to
low antimicrobial activity against the selected oral moderate antimicrobial activity when
pathogens when compared with 0.2% aqueous compared with standard antimicrobial agents
chlorhexidine. like chlorhexidine and tea tree oil.
Our results didn't support some previous 2. The different response of each microbial strain
to the various crude extracts indicated that
findings 16-20 that miswak extracts possess
each solvent extracted different chemical
considerable antimicrobial activity. By using
components of S. persica and that these
different antimicrobial assay in this study and
components either inhibited or enhanced
evaluating the effects under standardized test
the growth.
conditions, we have inferred that miswak extracts 3. The strength of the observed antimicrobial
manifested low antimicrobial activity. A case in activities may be due to the pH of the crude
point was the study conducted by Abo Al-Samh et extracts.
al.19 who used sodium hypochorite and compared 4. More studies performed by independent
it with miswak and discovered that the latter researchers using other techniques and
showed inhibition effects at much higher standardised test settings are needed to verify
concentrations than NaOCl. the previously published antimicrobial
The MIC chosen for the present study is similar results of S. persica extracts obtained with the
to that used by Cai et al.22 because similar agar diffusion test.
medium and wavelength were used. OD 650nm
value of 0.05 indicated no growth. Other studies References
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Geneva: WHO, 1987.
Many studies have been done to test the in 2. Wu CD, Darout IA, Skaug N. Chewing sticks:
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Azadirachta indica (Neem) and Salvadora persica Acknowledgement
(Arak) chewing sticks. Indian J Dent Res 1999;
10:23- 26. We extend our appreciation to Prof. Vidar Bakken
16. Almas K, Al-Bagieh NH. The antimicrobial effects o f and PhD student Ismail Darout for their technical
bark and pulp extracts of miswak, Salvadora support; to Prof. E. Al Bari, Dr. A. Alawid and Mr. B.
persica. Biomed Letters 1999; 60:71-75. Mohamed Ali for their assistance during the plant
17. Almas K, Al-Bagieh N, Akpata ES. In vitro identification and last but not the least, to Prof. S. Tegani
antimicrobial effects of freshly cut and 1-month-old and A. Hassan for their help in the scientific translation
Miswak extracts. Biomed Letters 1997;56:145-49. of the abstract.
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effects of aqueous and alcohol extracts of miswak Loan Fund for Education and the School of Medicine,
(chewing sticks). Cario Dent J 1997;13:221-24. University of Bergen, Norway.
19. Abo Al- Samh DA, Al-Bagieh NH. A study of the
antimicrobial activity of the miswak ethanolic extract

Saudi Dental Journal, Vol. 14, No. 1, January - April 2002