Influence of salvadora persica (miswak) extract on physical and antimicrobial properties of glass ionomer cement.
Abstract: AIM: To investigate physical and antimicrobial properties of Glass Ionomer Cement (GIC) combined with Salvadora Persica Extract (SPE). METHODS: SPE was added to GIC (Fuji IX) in concentrations of 1%, 2% and 4% w/w. The compressive strength and diametral tensile strength were measured at 1 h, 24 h and 7 days. The antimicrobial effect was tested in agar dilution assay in blood agar plates with Candida albicans, Streptococcus mutans, Streptococcus sanguis, Streptococcus mitis, Streptococcus salivarius and Actinomyces naeslundii as test organisms. GIC containing 5% chlorhexidine served as positive control. RESULTS: Significant differences were found for the compressive strength and diametral tensile strength as a result of adding SPE to GIC (p<0.05). GIC with 2 or 4 % SPE was significantly weaker than the GIC control, while GIC with 1% SPE was not different from the control. The mean values for the 4% SPE-containing specimens and the GIC control group ranged from 108.7MPa to 141.1MPa for CS and from 8.2MPa to 12.5MPa for DTS. The 1% SPE-containing specimens were not different in physical properties compared to the control GIC specimens; the 2% SPE-containing specimens were statistically slightly less strong (p<0.05), but within an acceptable range. As compared with pure GIC the antimicrobial properties of the SPE-containing specimens were increased significantly (p<0.01). It has been found up to a 2-fold increased inhibition compared to the GIC with increasing concentrations of SPE. For most microorganisms tested the SPE group inhibited less than Chlorhexidine, but significantly better than pure GIC (p<0.01). CONCLUSION: SPE could be a promising natural material as an additive to GICs. Further studies should include in vivo tests and other antimicrobial and physical properties of this combination.

Key words: salvadora persica (Miswak), glass ionomer, physical, antimicrobial
Article Type: Report
Subject: Cruciferae (Research)
Materia medica, Vegetable (Research)
Plant extracts (Research)
Authors: El-Tatari, A.
de Soet, J.J.
de Gee, A.J.
Shelib, M. Abou
van Amerongen, W.E.
Pub Date: 02/01/2011
Publication: Name: European Archives of Paediatric Dentistry Publisher: European Academy of Paediatric Dentistry Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 European Academy of Paediatric Dentistry ISSN: 1818-6300
Issue: Date: Feb, 2011 Source Volume: 12 Source Issue: 1
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: Netherlands Geographic Code: 4EUNE Netherlands
Accession Number: 277106722
Full Text: Introduction

Since the 1970s, when Glass Ionomer Cements (GICs) were introduced [Wilson and Kent, 1971], efforts have been made to improve the physical properties of those cements. Some of those efforts were by changing the powder to liquid ratios [Fleming et al., 2003], using different mixing methods [Nomoto et al., 2004] or using different curing methods [Kleverlaan et al., 2004]. Other studies dealt with the particle's size of the GICs and recently the effect of the environmental conditions on properties of the GICs was investigated [Algera et al., 2006].

Other aspects that have been the subject of investigation were improving the physical properties of the GICs by the addition of hydroxyapatite [Gu et al., 2005], boric acid [Prentice et al., 2006], phosphoric acid [Prentice et al., 2006a], oxalic acid [Prentice et al., 2006b] or lately casein phosphopeptide-amorphous calcium phosphate [Mazzaoui et al., 2003]. In the literature the addition of antimicrobial agents to GICs are mentioned. Examples are the addition of antibiotics such as metronidazole, ciprofloxacin and cefaclor [Pinheiro et al., 2005] that gave significantly greater antibacterial effect than conventional GIC. This however gives rise to possible antibiotic resistance and should therefore be avoided. Strontium showed good results against Actinomyces viscous [Guida et al., 2003].

Chlorhexidine when added to GICs gave good results from an antibacterial perspective in inhibiting the growth of a large collection of microorganisms [Botelho et al., 2004; Takahashi et al., 2006]. Studying the physical properties combined with the antimicrobial effect after adding those agents is a valuable approach, however the experimental GICs could not, up to now, be maintained to the ISO standard for their physical and mechanical properties. This is probably the reason why the combination of Chlorhexidine and other antibacterial substances with GICs has still not been incorporated into their production.

It is becoming more popular nowadays to introduce natural and herbal materials into dentistry. One of the most popular materials is the Salvadora Persica, a tree that is often used for making miswaks. Salvadora Persica is widely spread from West Africa to East Asia. The word Miswak is a general word referring to the wooden stick made from different plants. From some of these plants an extract has been added to tooth pastes and mouth rinses [Khalissi et al., 2004; Almas et al., 2005]. Salvadora Persica Extract (SPE) proved to have considerable antibacterial effect on some oral pathogens such as Candida albicans, Streptococcus mutans, Aggregatibacter actinomycetemcomitance, Lactobacillus acidophilus, Actinomyces naeslundii and Prophyromonas gingivalis [Abd El Rahman et al., 2002]. Chemical investigation of SPE has shown that it contains thymol and isothymol (both are well known antiseptic compounds), lignin, polysaccharides, derivatives of phenols, eugenol and eucalyptol [Al-Ali and Al-Lafi, 2002; Abd El Rahman, et al., 2003].

Recently a comparative study, using Chlorhexidine and SPE and their effects on human dentine have been investigated [Almas, 2002]. In that study the use of 50% SPE removed more smear layer and opened more dentinal tubules in the periodontally involved teeth than did Chlorhexidine.

As mentioned previously the addition of Chlorhexidine to GICs has been studied showing advantages (microbial inhibitory effect) and disadvantages (weak physical properties). However, the addition of SPE to GICs has never been investigated before. Therefore the aim of this study was to assess some of the antimicrobial and physical properties of a GIC when it is combined with SPE.

Materials and methods

Salvadora Persica Extract (SPE). Roots of Salvadora Persica roots that were at least 6 months old were brought from Saudi Arabia, chopped into small pieces and then ground in a blender (model No. TM-1273, Tomado Chopper). The chopped and blended SPE was then placed into a thimble, a thick filter paper tube with D x l = 35 x 150 mm and grade 603 (Carl Schleicher und Schull, Dassel, Germany), for extraction with methanol in a Soxhlet Extractor.

After extraction the methanol was removed by evaporation under vacuum using a rotary evaporator leaving a brownish oil. For purification a small amount of methanol was added just enough to dissolve the oil and to precipitate it again by adding diethyl ether. The precipitate and ether-methanol were separated by decantation. This procedure was repeated several times. Finally the SPE oily extract was kept in a closed flask in a refrigerator at 4[degrees]C.

Preparation of GIC and SPE mixtures. A conventional GIC, hand-mix version (Fuji IX, GC, Lot: 0509011, Exp: 2008-08), was used in this study. SPE oily extract was added to the liquid component of the GIC at weight percentages of 1%, 2% and 4% and hand mixed to homogeneous mixtures. Each mixture was then mechanically mixed with the GIC powder component in capsules (Maxicap, 3M ESPE) for 15 secs in a Silamat S5 Triturator (Vivadent, Schaan, Liechtenstein). The powder/liquid ratio was used as prescribed by the manufacturer.

Preparation of test specimens. After mixing the unaltered GIC (control material) and the GIC/SPE mixtures (experimental material) were injected into ten cylindrical holes of D x h = 4 x 5 mm in Teflon moulds, which were pre-warmed at 37[degrees]C. The moulds were covered on both sides by polyester strips and pre-warmed thick glass plates. Under these conditions the specimens were allowed to set for 10 mins after which they were removed from the moulds and stored in distilled water at 37[degrees]C until testing 1 hr, 24 hrs or 7 days after preparation. Malformed or large voids containing specimens were discarded, which left at least 8 suitable cylindrical specimens in each group.

Specimens for the antimicrobial tests were prepared using the same method, except the size was not the same because the mixtures were injected into pre-designed holes in blood heart infusion (BHI) agar plates, and were left to set for 2 hrs before replacing them onto blood agar plates that had been previously inoculated with specific microorganisms. For the antimicrobial tests an additional positive control group had been added by combing the GIC with 5% Clorhexidine.

Compressive Strength (CS) and Diametral Tensile Strength (DTS) determination. The CS and DTS were determined with a Hounsfield universal testing machine (Hounsfield, Redhill, S/N: 8702685, UK) at a crosshead speed of 0.5 mm/min. Also during the testing procedure the specimens were always kept wet with water. In total 210 cylinders were tested, 105 for the CS and 105 for the DTS.

Antimicrobial effect determination. The antimicrobial effect was tested in agar dilution assay in blood agar plates with Candida albicans, Streptococcus mutans, Streptococcus sanguis, Streptococcus mitis, Streptococcus salivaruis and Actinomyces naeslundii as test organisms. The bacteria were maintained on blood agar. Suspensions were made in BHI broth at a cell concentration of approximately 106 cells per ml. This was then spread onto blood agar plates. After allowing the plates to dry for 10 minutes, holes were punched at the size of the experimental GIC cylinders. The cylinders were placed into the holes. The testing plates were incubated at 37[degrees]C anaerobically with 80% N2, 10% CO2 and 10% H2 and after 5 days the diameters of inhibition zones formed around the specimens were measured using a caliper at three different points. Sizes of inhibition zones were calculated by subtracting the diameter of the specimen from the average of three measurements of the halo (the halo was oval or not exactly round in shape). Experiments without a growth inhibition by the GIC-Chlorhexidine positive control group were not used, considering that the GIC-Chlorhexidine as a golden standard in this experiment. The antimicrobial tests were repeated 5 times for each microorganism by using separate grown cultures.

Statistical analysis. A two-way ANOVA was performed to examine the effect of SPE and time (main effects) on the CS and DTS. Multiple pair-wise comparisons with the Tukey test at a p-level of 0.05 were performed to analyse differences within the subjects of the main effects. The microbiological inhibition tests were analysed for differences between groups by ANOVA and post hoc Tukey-Kramer multicomparison tests at a p-level of 0.01. The software used was SPSS 11.0 (SPSS Inc., Chicago, USA) and SigmaStat Version 3.0 (SPSS Inc, Chicago, USA).

Results

Sample size. A power calculation, made prior to the commencement of the study, indicated a sample size, to indicate a significant difference should have at least 8 specimens.

Compressive and Diametral Tensile Strength. The results for the CS and DTS and the statistical analysis are summarized in Tables 1 and 2. Significant differences were found for the CS and DTS for the two main effects, i.e. the addition of SPE [F(CS) = 11.7; F(DTS) = 12.0; p <0.001; Power (CS) = 1.00; Power(DTS) = 1.00] and time [(F(CS) = 70.5; F(DTS) = 8.9; p <0.001; Power(CS) = 1.00; Power(DTS) = 0.96]. GIC with 2 or 4% SPE was significantly weaker in CS and DTS than the GIC control, while GIC with 1% SPE was not different from the control. Furthermore the GIC with 4% SPE was weaker in CS and DTS than the GIC with 1% SPE. A significant increase in strength was observed for both the CS and DTS between 1 h and 7 days.

[FIGURE 1 OMITTED]

Antimicrobial effect. The results of the different specimens are shown in Figure 1. The inhibitory effect of the GIC-Clorhexidine on the different specimens indicated that all tests were valid. Statistical analyses showed that for all bacterial species, the SPE inhibited the growth stronger than the pure GIC (ANOVA for multiple comparisons). S. mutans and A. neaslundii were significantly inhibited with 1%, 2% and 4% SPE (p<0.01). The other bacterial species were inhibited at a lower level of significance for 1% (p<0.01). Between the 2% and the 4% a similar level of significance was found (p<0.01). The yeast C. albicans was only inhibited significantly with 4% SPE (p<0.01). It was observed that S. mitis and S. salivarius were significantly more sensitive for the combination GIC-SPE than the other bacterial species (p<0.01).

Discussion

GIC is often used as restorative material because of its relatively easy manipulation, self-setting characteristics, adhesive properties and its relative resistance to the effects of saliva. Furthermore GIC has been proven to show antimicrobial properties against several microorganisms, possibly, due to fluoride release [Herrera et al., 1999; Massara et al., 2002], However, this phenomenon has been disputed in other studies showing that the amount of fluoride released from GIC is not sufficient to show an antibacterial effect against S.mutans [Mazzaoui et al., 2000]. Therefore the aim of the present study was to modify the GIC (Fuji IX) with additional antimicrobial substances. Nevertheless it is necessary that any modification should not lead to inferior physical and mechanical properties of a restorative material. That is why this study started with a physical analysis of the SPE-modified GIC. Because it is difficult to prepare SPE and to manipulate experimental GICs specimens (due to the risk of dehydration), a decision was made to investigate the most common physical properties mentioned in the literature; the compressive strength (CS) and the diametral tensile strength (DTS) [Xie et al., 2000; Yap et al., 2003; Eduardo et al., 2004].

In this study it has been found that adding 1% SPE to the GIC did not change the physical properties significantly. Moreover the addition of 2% and 4% SPE to the GIC did not result in material distortion. The physical properties were comparable to other commercially available GICs [Eduardo et al., 2004]. Based on these results we concluded that GIC (Fuji IX) could be modified with SPE up to 4% without dramatically changing its physical properties.

Clorhexidine has been added to GIC in several studies. In our search modified GIC containing 5% Clorhexidine was used only as a positive control and found it to be effective against the whole panel of microorganisms used. This study showed that the antimicrobial properties increased significantly by adding higher concentrations of SPE. This was most prominent for S. mutans and S. sanguis, where they were almost not affected by the pure GIC. This is an important issue as S. mutans has been associated with secondary caries in many studies [Thomas et al., 2008]. The S. mitis and S. salivarius were affected significantly in our study, but that would not affect our aim, because those organisms are well known as normal oral flora.

Candida albicans was used in this study because it has been strongly associated with secondary and dentine caries [Thomas et al., 2008]. The addition of SPE was only significantly effective against the Candida albicans at 4% concentration; it was even more effective than the experimental Clorhexidine-GIC group. This means that for effective antimicrobial properties it is suggested that 4% SPE be used in modifying the GIC. Whether this is also needed in vivo should now be evaluated in clinical studies.

Although the GIC (4% SPE) specimens showed lower values for the CS and the DTS compared with the GIC control, it could still be investigated to be used in less stress bearing applications such as in Class V or as a temporary filling material in some techniques where complete caries removal is not recommended such as the stepwise excavation or the indirect pulp capping techniques.

This in vitro study is the first step in modifying a filling material by adding a natural product. The purification of SPE is an aspect that should be further investigated, as the brownish oil we have used in this study was not a pure substance. Occasionally we were able to isolate a crystalline product, which may have been the pure form of SPE. However the amount was too small to carry out all the experiments for this study. It is obvious that pure SPE will change the results in a positive way.

Conclusion

Based on the tests that herein it is concluded that the addition of 1% SPE maintained the levels for the CS and the DTS, and the addition of 4% SPE, although decreasing the physical properties of the GIC, gave promising results in the antimicrobial tests especially regarding the S. mutans, the S. sanguis and the Candida albicans. Further studies, using SPE, are recommended to investigate higher purification levels, other physical properties, such as the setting time and bond strength to dentine and enamel, measure the fluoride release and mineralizing effect, to evaluate other filling materials or luting cements with the SPE and in-vivo behaviour after a suitable balanced amount of SPE has been found and also to assess the preventive effect when combined with different fissure sealant materials, based on resin as well as on glass ionomer cements.

Acknowledgments

The authors wish to acknowledge the support of GC Europe for providing the Fuji IX glass-ionomer cement.

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A. El-Tatari *, J.J. de Soet **, A.J. de Gee ***, M. Abou Shelib ***, W.E. van Amerongen *

Depts. of Paediatric Dentistry *, Oral Microbiology ** and Dental Material Science ***, Academic Centre of Dentistry Amsterdam (ACTA), The Netherlands.

Postal address: Dr. A. El-Tatari. Dept of Cariology, Endodontology & Pedodontology, ACTA. Gustav Mehlerlaan 3004, 1081 LA, Amsterdam, The Netherlands.

Email: ahmed.eltatari@gmail.com
Table 1. Mean compressive strength and standard deviation of
unaltered GIC (control) and GIC altered with Salvadora Persica
(Miswak) Extract on physical and antimicrobial properties of Glass
Ionomer Cement with 1, 2 and 4% SPE measured 1h, 24 h and 7
days after mixing.

Time          GIC             GIC             GIC            GIC
           (control)       (1% SPE)        (2% SPE)       (4% SPE)

1 h          111.3           105.0           95.5           72.6
          (25.6) (a)      (7.0) (a) |     (5.4) (a,b)     (6.6) (b)

24 h         150.5           118.1           123.8          111.9
         (22. 5) (a) |   (19.0) (b) |   (23.1) (a,b) |   (12.5) (b)

7 d          164.1           182.9           146.7          141.7
        (26.2) (a,b) |    (32.7) (a)     (41.4) (b) |    (22.9) (b)

Same superscript letters indicate no significant difference between
data in rows (p > 0.05) and vertical lines indicate no significant
difference between data in columns (p > 0.05).

Table 2. Mean diametral tensile strength and standard deviation
of unaltered GIC (control) and GIC with 1, 2 and 4 % SPE measured
1h, 24 h and 7 days after mixing.

Time           GIC                  GIC
            (control)            (1% SPE)

1 h     12.1 (2.2) (a) |    10.3 (3.1) (a,b) |
24 h    11.9 (3.7) (a) |     9.7 (2.6) (a,b) |
7 d     13.6 (3.1) (a) |      13.1 (3.9) (a)

Time            GIC                 GIC
             (2% SPE)             (4% SPE)

1 h      8.9 (2.3) (a,b) |    7.1 (1.5) (b) |
24 h     8.8 (2.0) (a,b) |    7.8 (2.0) (b) |
7 d     11.6 (3.7) (a,b) |    9.5 (2.5) (b) |

Same superscript letters indicate no significant difference between
data in rows (p > 0.05) and vertical lines indicate no significant
difference between data in columns (p > 0.05).
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