Indian Journal of Pharmacology 2000; 32: 11-14
RESEARCH PAPER
ANTIMICROBIAL ANIONIC COMPONENTS IN MISWAK EXTRACT
IDENTIFICATION AND QUANTIFICATION OF SOME POTENTIALLY ANTIMICROBIAL ANIONIC COMPONENTS IN MISWAK EXTRACT ISMAIL A. DAROUT1, ALFRED A. CHRISTY2*, NILS SKAUG1, PER K. EGEBERG2 1Department 2Department
of Odontology, Faculty of Dentistry, University of Bergen, Bergen, Norway. of Chemistry, Agder College, Kristiansand, Norway.
Manuscript Received: 6.10.99 SUMMARY
Revised: 15.11.99
Accepted: 27.12.99
Objective: To identify and quantify some potential antimicrobial anionic components in Salvadora persica root and stem aqueous extracts. Methods: Extraction of powdered root and stem samples was performed by soaking the powder in sterile de-ionised distilled water for 24 h at 4 oC. Each 100mg of the freeze-dried extract was reconstituted with 10 ml de-ionised distilled water and filtered through a 0.45 µm cellulose acetate filter. The anionic components of the filtered extracts were identified and quantified by capillary electrophoresis. Results: The root and stem extracts contained chloride, sulphate, thiocyanate and nitrate in the following concentrations (w/w %) in stem and root extracts, respectively: 6.84% and 4.64%, 20.1% and 19.85%, 0.38 and 0.28%, and 0.05% and 0.05%. Only the differences in chloride were statistically significant (p< 0.05). Conclusion: S. persica contains potential antimicrobial anionic components and that capillary electrophoresis is a convenient method for their identification and quantification.
KEY WORDS
Salvadora persica
miswak extracts
anti-microbial anionic components
INTRODUCTION Miswak is a chewing stick prepared from the roots, twigs or stems of Salvadora persica L. (Salvadoraceae) and widely used in Middle Eastern and Eastern African cultures. It has been shown that extracts of miswak posses various biological properties including significant antibacterial1 and anti-fungal effects2. Extracts of S. persica and other related plants may be effective against the bacteria that are important for the development of dental plaque. Therefore, it has been claimed that miswak sticks may have antiplaque effects and may also affect the pathogenesis of periodontal diseases by reducing the virulence of periodontophathogenic bacteria3. The anti-microbial and cleaning effects of miswak have been attributed to various chemicals detectable in its extracts. These effects are believed to be due to its high content of sodium chloride and potassium chloride as well as salvadourea and salvadorine,
capillary electrophoresis
saponins, tannins, vitamin C, silica, and resin4, in addition to cyanogenic glycoside and benzylisothio-cyanate. Thus, it has been reported that some anionic components naturally occurring in plant species exert anti-microbial activities against various bacteria. Nitrate (NO3-) has been reported to affect active transport of porline in Escherichia coli as well as aldose from E. coli and Streptococcus faecalis 5. In addition, it has proved to inhibit active transport oxidative phosphorylation and oxygen uptake by Pseudomonas aeruginosa and Staphylococcus aureus 5. Other anti-microbial anionic components present in some plant species include sulphate (SO42-), chloride (Cl-) and thiocyanate (SCN-). SCN- acts as a substrate for lactoperoxidase to generate hypothiocyanite (OSCN-) in the presence of hydrogen peroxide. OSCN- has been demonstrated to react with sulfhydryl groups in bacterial enzymes which in turn leads to bacterial death. Acid production in human dental plaque in vitro has been reported to be
Correspondence: Alfred A. Christy, E-mail:
[email protected]
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ISMAIL A. DAROUT et al.
inversely proportional to the concentration of OSCNin the test system, while supplementing saliva with hydrogen peroxide and SCN- inhibited acid production6. Despite the wide use of miswak and its containing products, its chemotherapeutic value has not been fully substantiated and the mode of action of its bioactive compounds against oral bacteria has not yet been established. Therefore, the aim of the present study was to identify and quantify some potential anti-microbial anionic components in S. persica root and stem aqueous extracts by using capillary electrophoresis techniques. MATERIALS AND METHODS Collection of plant materials: The stems and roots of S. persica were collected from the Khartoum botanical garden, Khartoum, Sudan, as authenticated by Dr Ikhlas A/Bari, Department of Botany, Faculty of Science, University of Khartoum. The collected plant materials were dried at room temperature for 10 days and then shipped to the Laboratory of Oral Microbiology, University of Bergen, cut into small pieces and powdered using an Omni mixer (MFRS and Distrs, Conn. USA). Aqueous extraction and calculation of yields: Twenty grams of the powdered root or stem were transferred to sterile wide-mouthed screw-capped bottles of 200 ml volume. 100 ml of sterile de-ionised distilled water was added to the powder samples which were allowed to soak for 24 h at 4°C. The mixtures were then centrifuged at 2000 rpm for 10 min. at 4°C. The supernatants were filtered through a 0.45 µm membrane (Millipore Corp., Bedford, MA, USA) and freeze-dried. The yields of root and stem extracts were calculated as the amount of freezedried extract expressed as weight percentage of the plant material used for extraction (w/w%). Reconstitution of freeze-dried extracts and capillary electrophoretic analysis: One-hundred mg of freeze-dried root or stem extracts was reconstituted with 10 ml de-ionised distilled water. The capillary electrophoretic analysis of the root and stem reconstituted extracts was then carried using a Hewlett Packard HP3D apparatus. A standard solution containing bromide Br-, Cl-, SO42-, NO3- and SCN- was first analysed and the migration times and peak ar-
eas of its anions were compared with those of the anions of solutions of root or stem reconstituted extracts. They were further confirmed by spiking the sample with relevant anions. Quantification of the anions was carried out using calibration standards. Statistical analysis: The 't ' test for paired samples and simple regression analysis were used to evaluate statistical differences and the association between the concentrations of different anionic components and corresponding peak areas. P values
