Maxillofacial silicone prostheses are fundamental in rebuilding facial aesthetics and restore function for patients with facial trauma, congenital deformities, or post-surgical interventions [1,2]. Guaranteeing the durability and well-being of wearers demands proper care with disinfection being a key factor to prevent microbial colonization and sustain hygiene [3,4]. 

One of the most serious problems associated with application of the silicone prosthesis is microbial colonization on its surface with bacteria, fungi and plaque which end with infection and/or inflammation of the mucosa [5,6]. Recently, due to widespread antimicrobial drugs intake around the world which lead to emergence of resistance microbial species and under high demand to discover new antimicrobial agent a medicinal cost plant may be the solution [7]. Medicinal plants are safe, effective, and economic so that many of plants extract may be act as natural substitutes to synthetic medicines [8,9].

Natural medicinal plants extracts are widely used as antibacterial (bactericidal, bacteriostatic), antifungal, antioxidant, anti-inflammatory and anti-tumor. These extracts have not the ability to develop bacterial resistance and it has a great benefit over the synthetic products [10,11].

In recent years, there has been a burgeoning interest in natural and herbal alternatives for disinfecting medical devices, including silicone-based prostheses. Noteworthy among these alternatives is Salvadora persica L., commonly known as Miswak or Siwak, exhibiting promising antimicrobial properties. Salvadora persica L., a traditional chewing stick, is recognized for antibacterial and anti-inflammatory properties attributed to its bioactive constituents [12,13]. Salvadora persica L. root sticks are commercially accessible, securely packaged in sealed wraps, and are readily found in local shops. These root sticks have been manufactured in Saudi Arabia and originate from the Salvadora persica L. plant, belonging to the Salvadoraceae family. The roots of Salvadora persica L. serve various purposes and are available for purchase, contributing to the traditional use of this plant in oral hygiene practices and other applications [13].

An aqueous extract mixture of Salvadora persica L. and green tea was used as a potent antibacterial herbal medicament and successfully incorporated into heat cured acrylic based denture soft liner to obtain a material with a continuous drug-delivery system against Staphylococcus aureus [14]. Salvadora persica L. also incorporated into glass ionomer cements and showed significant reduction in bacterial growth [15].

2.1 Materials used in the study

Materials used are listed in (Table 1).

Table 1: Materials used in the study.

2.2 Salvadora persica L. extraction

The extraction process of Salvadora persica L. root sticks involved the following steps: First, the root sticks were meticulously cut into small pieces (1 cm) using sharp scissors. Then, the small pieces were further reduced in size by hammering with a hammer and the resulting pieces were ground into fine fragments by the use of an electrical grinder at speed 5 and a duration of 10 minutes. This mechanical processing aimed to enhance the contact surface area between the plant material and the solvent, consequently expediting the rate of the extraction process [16].

Following the preparatory steps, 100 grams of the finely ground S. persica L. powder was introduced into 1000 milliliters of deionized distilled water for the maceration process, maintaining a mixing ratio of 1 gram of plant material per 10 milliliters of solvent. The aqueous extracts obtained were subjected to a filtration process using filter paper to eliminate coarse plant particles. The filtrate was then subjected to centrifugation at 10,000 revolutions per minute for 10 minutes, followed by an additional filtration step using filter paper (JIAO JIE, China). The resulting filtered extract underwent drying in an oven set at 40°C for 3 hours to yield dry S. persica L. powder.

Maceration was selected as an appropriate technique for Salvadora persica L. raw material plants extraction. The main concept of maceration is the separation of soluble plant component obtained from rigid plant bundle through soaking in a suitable solvent at room temperature [16,17].

S. persica L.  powder was then milled by using a planetary ball milling machine (Yangzhou Nuoya Machinery Co., Ltd., China) for 90 minutes (15 min. operation followed by 15 min. shutdown to prevent heat generation) at a speed of 300 rpm. The machine consists of hollow cylindrical pots rotating around their axis and partially filled with milling balls made from ceramic. It relies on the energy released from the impact and attrition between the balls and powder of the material to be milled. The milling time of 90 min was selected based on a trial stage of different milling times (30 min, 60 min, and 90 min.), and the size was verified each time with the particle size analyzer.

2.3 Particle size analysis

Laser diffraction (LD) particle size analyzer was used to determine the size of powder after milling. Ethanol was used as a dispersant. The LD method involves the detection and analysis of the angular distribution of scattered light produced by a laser passing through a dilute dispersion of the particles [18]. Particle size for S. persica L. ranged from 312.51 to 447.72 nm.

2.4 Specimen grouping

In this in-vitro prospective study, specimens grouping involved 120 silicone specimens (40 for bacterial test, 40 for hardness test, and 40 for roughness test) were utilized in this study. The specimens were divided into four groups (10 specimens for each group) based on the concentration of powder mixed with silicone (0% Control, 0.5%, 1%, and 1.5%).

2.5 Acrylic molds preparation

AutoCAD 2015, developed by Autodesk Inc. in San Rafael, CA, USA, was employed for creating the dimensions, following which a laser cutting device was utilized (JL,1612, Jinan Link Manufacture and Trading Co., Ltd., China) to cut the acrylic sheets (Perspex Cell Cast Acrylic, Clairvaux les Lacsrance, France) according to each test. The mold consists of three parts which are the base, the mold space into which silicone was poured, and the cover. Additional equipment includes G, clamps, nuts, and screws to further secure the mold parts together.

2.6 Mixing

As directed by the manufacturer; the mixing ratio was 10 of part A (base) to 1 of part B (catalyst) by weight, the manufacturer also recommends using vacuum mixer or vacuum chamber (Figure 1) to reduce air entrapment which will affect the mechanical properties. In order to get the best results of mixing and curing, the process should be done at 23° C and 50% relative humidity [19]. The samples with added powder were prepared by adding the powder in three different concentrations (0.5%, 1%, and 1.5% by weight) to silicone material. The weight of the added powder was subtracted from the weight of the base (part A) to obtain accurate base to catalyst ratio.

Figure 1: Mixing chamber.

2.7 Preparation and storage of specimens

Specimens were prepared by coating the mold cover with two layers of alginate solution as a separating medium, followed by slowly adding silicone mixture to fill all sample spaces, slightly overfilling to ensure completeness. The top section of the mold was then assembled over the matrix part using moderate hand pressure, screws, nuts, and G-clamps to eliminate air bubbles and excess silicone. Polymerization occurred over 24 hours at room temperature (23±2°C), after which samples were carefully removed from the mold. Cleaning involved rinsing under tap water, drying with paper towels, and fine-tuning with a scalpel to remove surplus material [20].

2.8 Muller Hinton Agar preparation

In accordance with the manufacturer's protocol, Muller Hinton agar was meticulously prepared by dissolving 38 grams of the medium in 1000 milliliters of distilled water. After dissolution, the solution underwent boiling for a duration ranging from one to ten minutes to ensure complete homogenization. Following this phase, the solution was subjected to sterilization at 121°C and 15 pounds per square inch (PSI) for a duration of 15 minutes using an autoclave device (Tuttnauer, USA), thereby achieving sterility. Subsequently, the solution was allowed to cool to room temperature. The cooled agar was then carefully poured inside sterile petri-dishes (China) on horizontal surface to ensure that the layer is consistent and have a uniform depth.

Stringent measures were then taken to verify that the final pH of the agar medium was maintained at 7.3 ± 0.1 when measured at 25 ºC. Lastly, the prepared agar plates were stored at temperatures ranging from 2-8 ºC following to the guidelines specified by the Clinical and Laboratory Standards Institute [21]. 

2.9 Isolation and identification of Staphylococcus aureus
Sterile cotton swabs used to collect culture specimens of S. aureus from the throats and saliva of patients. A total of 50 specimens obtained from patients attending the Department of Educational Laboratories at Tikrit hospital in Tikrit/ Iraq. The specimens were then inoculated into blood agar which is a suitable growth medium for S. aureus. The preparation of blood agar followed manufacturer recommendations. The agar plates containing the specimens were then incubated in aerobic conditions for a duration of 48 hours at 37°C, following guidelines set by the Clinical and Laboratory Standards Institute [21]

The identification of Staphylococcus aureus was based on characteristic features. S. aureus typically manifests as light golden circular colonies on blood agar (Figure 2). Identification methods included catalase testing, stain methods, and coagulase tests. The catalase, stain, and coagulase tests collectively aid in confirming the presence of S. aureus.

Figure 2: Staphylococcus aureus on blood agar.

2.10 Disc diffusion test

Disk shaped specimens (5 mm diameter and 2 mm thickness) were used. The Kirby-Bauer method, as recommended by the World Health Organization (WHO) in 2003, was employed to assess the antimicrobial efficacy of the Salvadora persica L., incorporated into silicone specimens. The bacterial suspension for testing was prepared by selecting 1,2 isolated colonies of Staphylococcus aureus from the incubated culture. These colonies were then placed into a test tube containing 4 milliliters of normal saline, resulting in a bacterial suspension with a turbidity approximately equivalent to 1.5x10^8 CFU/mL.

Subsequently, a sterile cotton swab was utilized to retrieve a small portion of the bacterial suspension, which was then evenly spread on Mueller-Hinton agar medium through careful streaking on the media surface. Following a 10,minute incubation period, specimen discs were introduced onto the agar using sterile forceps, ensuring proper and firm contact with the agar surface. The plates then inverted and incubated for 18-24 hours at 37 ºC. 

2.11 Shore A hardness

Shore A hardness samples were fabricated according to ISO 7619,1 (2010) specifications. Each sample measuring 40mm in length, 40mm in width, and 6mm in thickness. These dimensions were chosen to ensure adequate outer surface area for conducting five measurements using a shore A durometer (Ezitown, China) as shown in figure 3, with a 6mm distance between each measurement point. Additionally, a 12mm distance from the sample margin was maintained to facilitate accurate hardness testing in accordance with the standard. The mean of the five hardness readings was subsequently calculated for each sample.

Figure 3: Shore A durometer for hardness test.

2.12 Roughness

The samples used for surface roughness testing had the same dimensions as those used for Shore A hardness tests. A profilometer device was employed (Figure 4). This device features a stylus that is moved over the surface of the sample, and three readings are recorded for each sample following the device's instructions. The average value is then considered as the roughness.

Figure 4: Surface profilometer.

3.1 Disc diffusion test

The results of disk diffusion method of group D specimens exhibited the highest mean value of inhibition zone as (16.6760 mm), followed by group C specimens (14.1643 mm), while the lowest value (12.9520mm) was obtained in group B specimens as presented in figure and table 2. Regarding group A (Control group), no inhibition zone was obtained (Figure 5 and 6).

Figure 5: Bar chart of disk diffusion test representing the means and standard deviation.

Figure 6: Disk diffusion method results of a) Group A specimens (Control); b) 0.5% Salvadora persica L.; c) 1% Salvadora persica L.; d) 1.5% Salvadora persica L. specimens.

There was highly significant difference between groups A and C, also highly significant difference between groups B and C (Table 2).

Table 2: Descriptive statistics and one-way ANOVA test for disk diffusion test.

All experimental groups demonstrated a slight decrease in hardness after the addition of powder compared to the control group, however the decrease was non-significant as revealed by one-way ANOVA test (Figure 7, Table 3).

Figure 7: Bar chart of Shore A hardness test representing the means and standard deviation.

Table 3: Descriptive statistics and one-way ANOVA test for shore A hardness.

3.3 Roughness

The results revealed an increase in surface roughness for all groups after the addition of powder compared to the control group (Figure 8 and Table 4).

Figure 8: Bar chart of Shore A hardness test representing the means and standard deviation.

There was a highly significant difference between groups (A) and (C), (A) and (D) only. No difference between the remaining (Table 4).

Table 4: Descriptive statistics, one-way ANOVA, and Tukey HSD test for surface roughness.

The outcomes of the disc diffusion test in this investigation revealed a notably significant increase in the inhibition zone surrounding silicone specimens infused with Salvadora persica L. Powder compared to control specimens (P<0.001). This enhancement may be attributed to the presence of phytochemical constituents in Salvadora persica L. known for their potent antibacterial properties, including saponins, cardiac glycosides, flavonoids, alkaloids, Benzylisothiocyanate, nitrates, sulfur, tannins, trimethylamine, salvadorine, various phenolic compounds, chloride, and steroids [22-24].

In addition, saponins and alkaloids, prevalent in plants and primarily glycosides, harbor antibacterial compounds capable of disrupting microbial DNA 25. Additionally, hydroxylated phenolic compounds, known as flavonoids, play a crucial role in combating bacterial infections by interacting with bacterial cell walls and proteins, corroborating the observed inhibition zone around heat-cured soft-liner specimens infused with Salvadora persica L. [26,27]

Additional support for the antimicrobial efficacy of Salvadora persica L. comes from the role of Benzyl isothiocyanate, a compound found within the extract, in combating viral, microbial, and bacterial infections. Through a hydrogen peroxide antimicrobial system, Benzyl isothiocyanate can disrupt the functionality of cytoplasmic membranes of the bacteria which results in an inhibition of the glucose transport and subsequent leakage of amino acids, peptides, and potassium ions. Such disruption inside the bacterial cell leads to the inhibition of bacterial glycolysis and cell death [28,29].

 Top of Form

Salvadora persica L. has been studied extensively for its antimicrobial properties, which have been confirmed by different studies. These properties are attributed to elements such as nitrates and sulfates, which can hinder oxygen consumption and phosphor-oxidative transport activity of S. aureus [30-32].

 Top of Form

Another important factor that contributes to the antimicrobial activity is the fluoride content in Salvadora persica L., which demonstrates a strong antibacterial properties by interacting with intracellular polysaccharides and inhibiting glycolytic enzymes in the bacteria [33].

The soft nature 

The observed decrease in hardness and increase in surface roughness upon the addition of Salvadora persica L. into A-2186 RTV silicone can be attributed to the softer nature of phytochemical constituents including sulfur and cardiac glycoside derivatives which disrupts the cohesive forces inside the silicone matrix, resulting in a decrease in material hardness. This disruption happens at the atomic level affecting the orderly arrangement of atoms and thereby decreasing the overall resistance of material to indentation [34,35]. Additionally, the addition of such soft elements into the silicone matrix can create local variations in material properties. This leads to uneven surfaces and an increase in surface roughness. These variations arise because of differential interactions between the silicone polymer chains and the phytochemical compounds, resulting in localized deformations on the surface of the silicone. Moreover, changes in the molecular structure and interactions inside the silicone matrix caused by the presence of Salvadora persica L. can further intensify surface roughness by the formation of surface projections or irregular patterns [35,36]. Such changes can arise from shifts in molecular mobility, cross-linking density, or surface energy within the material, which together participate in the observed increase in surface roughness. 

The addition of Salvadora persica L. into maxillofacial silicone material has demonstrated promise in improving the antibacterial efficacy. This improvement has been observed along with a slight increase in surface roughness, which remains within acceptable measures, as well as a decrease in material hardness.

Ethical approval

An In-vitro study

Conflict of interest

None.

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