Antibacterial Activity of Salvadora persica L. Ethanol Extracts against Root Canal Pathogens from Clinical Isolates

Muhammad Musthafa Poyil^1*, Ponmurugan Karuppiah^2,3 and KP Shamna⁴

^*Correspondence: Muhammad Musthafa Poyil, Department of Basic Medical Sciences, College of Medicine, Prince Sattam Bin Abdulaziz University, Saudi Arabia, Email:

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Introduction

Decaying of teeth has been listed as a chronic infection that occurs commonly around the globe. The situation is severe in developing countries as 60–90% of schoolchildren and most of the elders are affected by this disease [1]. The odonto-periopathic bacteria present in the plaque are the primary cause of these dental infections. Reducing the number of bacteria or preventing them completely is vital in tackling these infections [2,3]. Despite the effectiveness of commonly used antibiotics like erythromycin, penicillin etc. in preventing dental caries in animals and humans [4], their side effects including supra-infections, teeth staining and hypersensitivity reactions make them less prevalent in the treatment [5,6]. Also, the emergence and spread of multidrugresistant (MDR) bacteria due to the uncontrolled use of antibiotics, the need for the search of new antibacterial agents from the nature is highly demanded [7]. This is how the researchers have begun to concentrate on plant-based bioactive compounds which are effective and at the same time economic with no remarkable side effects.

Since time unknown, the use of plants and other natural substances in dental care and in the overall cleanliness of the oral cavity has been practiced worldwide and recorded [8]. Researchers have concluded that the toothbrushes used in the modern period should have been developed from the plant sticks chewing and ‘tooth picking’ habit practiced by Babylonians since 3500 BC as mentioned in Roman and Greek records [9]. Even today, the herbal sticks with a high degree of antimicrobial potentials are being used by many of the communities in oral hygiene and as a remedy for many of the dental diseases [10]. Various species of plants are being used to prepare chewing sticks in the Middle East, Africa, Asia and South America [11].

It has been estimated that there are 182 plants which can be used in brushing the tooth and the stick widely known as the "miswak," taken from Salvadora persica is considered as the most commonly used one [12]. Salvadora persica grow as a shrubs or small trees with trunks less than one foot in diameter. The sticks have an attractive fragrance with a pungent taste. The bark of this plant is cracked and the extremities are pendulous and whitish in color [13]. The plant is seen in different parts of the world Nepal, India, Pakistan, Malaysia, Iran, Iraq, Saudi Arabia and in African countries [14]. Most parts of the plant including the stem and roots are used in oral hygiene [11] including the toothpicks made by smaller sticks [15]. Several studies using the aqueous extract from the plant have detected the presence of components with antibacterial activities against periodontal and cariogenic pathogens including Staphylococcus aureus, Streptococcus mutans, Streptococcus salivarius and anaerobic Streptococci [16,17]. Underlining its importance in oral health, the WHO-World Health Organization has encouraged and recommended the Miswak-Salvadora persica sticks chewing in the different parts of the globe where it is traditionally used for the dental care and overall buccal hygiene [16].

Chemically, the Salvadora persica has various potential organic and inorganic compounds assisting in oral health maintenance like antibacterials, antiviral, tooth strengthening agents, anti-carcinogens, anti-genotoxins, abrasives, coating agents etc. [18,19]. The primary dental caries causing bacteria, dominating in most of the cases have been identified as Streptococcus mutans and Lactobacillus sp. and having acidogenic and uricabilitic properties which are the determining factors in dental caries formation they ensure an acidic environment along with some other acidophiles [18,20-23]. The biofilm formation by these bacteria and other species thereby developing into a matrix in dental plaques is another major issue. The lactic acid production as a result of carbohydrate fermentation by many agents including Streptococcus mutans, Candida albicans and Lactobacillus sp. deteriorate the scenario resulting in total destruction of the tooth surfaces [24,25]. Dental caries may lead to further complications as the buccal bacterial flora can even promote carcinogens. Cultureindependent methods have elucidated a much greater complexity of oral microbial flora promoting carcinogens by the cumulative activities of the members of the community [24]. The present study examines the chemical components present in the Salvadora persica extracts and their effect on selected, clinically isolated root canal bacterial pathogens using different methods.

Experimental

Collection and extraction of plant materials

The healthy plant root samples of Salvadora persica were collected from the medicinal plant markets in India and were identified by K. Rajarathinam, Department of Botany, V.H.N.S.N. College, Virudhunagar, Tamil Nadu, and India. Surface washed roots were cut into small pieces with the help of a sterile knife. Then it was powdered by a blender. This powder was subjected to the extraction. The ethanol extraction of Salvadora persica root samples was done using the Soxhlet apparatus standard procedure suggested by Anees et al. [26].

Test microorganisms

The most common root canal disease causing bacteria were collected from Sun Micro Laboratory, Erode, Tamil Nadu, and India. Also, reference strains Streptococcus mutants (MTCC 890), Staphylococcus aureus, Lactobacillus acidophilus (MTCC 447), Actinomyces sp. were obtained from MTCC, Chandigarh, India.

Screening of antibacterial properties of salvadora persica ethanol extracts

Agar well diffusion

At a cell density of 1 X 108 CFU/ml (in accordance with the 0.5 McFarland standards), the broth culture of the selected bacteria was prepared. Using a sterilized cotton swab, the aliquot was spread onto Muller Hinton agar. The plates were dried at room temperature by keeping for 30 minutes [27]. Using a sterile cork borer, wells with a diameter of 6mm were made on the plates keeping a 2mm from the plate edge. The leaf extracts at a volume of 50μl were added aseptically into agar well. The positive controls were 30 μg/ml of chloramphenicol. The negative controls were the extraction solvents, viz ethanol, ethyl acetate and hexane. The agar plates were kept for about 40 minutes on the working table for solidification. The plates were then incubated at 37oC for 24 hours. The tests were conducted in triplicates. A zone of inhibition with ≥ 7mm diameters formed surrounding the wells was considered as the sensitivity of the organisms to the extract [28].

Minimum inhibitory concentration (MIC) determination

The extracts which produced an inhibition zone with ≥ 7 mm diameter were selected to find the MICs by agar well diffusion methods. For this, the serial dilutions of the extracts (500 μg/ml) were prepared as 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256 to bring 250 μg/mL, 125 μg/mL, 62.5 μg/mL, 31.25 μg/mL, 15.63 μg/mL, 7.81 μg/ mL, 3.95 μg/mL and 1.95 μg/mL concentrations respectively. The wells were filled with the extracts aseptically. After incubating at 37oC for 24 hours, the zones inhibition was measured. The lowest concentration at which the growth inhibited was recorded as the MIC for the sample.

Phytochemical screening of the extracts

A preliminary screening test for certain phytochemical components like carbohydrates, glycosides, alkaloids, fixed oil and fats, phenolic compounds, phytosterols, flavonoids, proteins and amino acids, saponins, lignin, gum and mucilage etc. was performed using standard protocols.

Results

The findings of this investigation proved that Salvadora persica root ethanol extract is an effective tool for inhibiting the tested root canal bacteria such as Staphylococcus aureus, Streptococcus mutants, Lactobacillus acidophilus and Actinomyces sp. The inhibition response of Salvadora persica in the agar well diffusion method on the selected root canal bacteria is displayed in Table 1 and Figure 1. The ethanol extracts showed inhibitory activity with greater effectiveness against most of the eight microorganisms used in this investigation, followed by acetone and other hexane extracts. Among the root canal clinical pathogens Staphylococcus aureus and Streptococcus mutants were the most susceptible, followed by Lactobacillus acidophilus and Actinomyces sp., which were the least inhibited by the Salvadora persica ethanol extracts. The diameter of zone inhibition varied between 8.5 mm and 17.6 mm (Table 2 and Figure 2). The ethanolic extract of Salvadora persica showed the highest zone of inhibition of 17.6 mm against Streptococcus mutants Strain 1, followed by the Staphylococcus aureus Strain 2 (16.7 mm); the lowest inhibition zone exhibited against Actinomyces sp. (8.3 mm).

Table 1: Antibacterial activity of Salvadora persica extract against bacterial strains.

Table 2: Antibacterial activity of the Salvadora persica extract against dental caries causing bacterial strains.

Figure 1. Antibacterial activity of extract of the Salvadora persica against bacterial strains.

Figure 2. Antibacterial activity of the Salvadora persica extract against dental caries causing bacterial strains.

The MIC values of crude ethanolic extracts were 31.25, 62.50, 125, 250, 500 μg/ml against S. mutants Strain 1 and 3, S. aureus Strain 2, S. mutants Strain 4, L. acidophilus and Actinomyces sp. respectively (Table 3 and Figure 3). The MICs were as low as 31.25 μg/ml of extracts against root canal bacteria and underline the presence of potential bioactive compounds in the ethanolic extracts. Hence, it can be suggested that the ethanolic extracts of Salvadora persica may have molecules with potential activities for treating dental caries caused by pathogenic bacteria.

Table 3: MIC value of ethanolic extracts of Salvadora persica.

Figure 3. MIC value of ethanolic extracts of Salvadora persica.

Thus, as shown above, the preliminary investigation for the presence of potential phytochemical compounds in the root extracts of Salvadora persica showed the activity of various chemical constituents, the majority of which are effective antimicrobial compounds. This work confirmed the presence of glycosides, carbohydrates, phytosterols, alkaloids, saponins, lignin, phenolic compounds, flavonoids, tannins, proteins and amino acids, fixed oils and gum and mucilage.

Discussion

The findings of this investigation are underlined by a couple of studies conducted by various researchers. For example, Almas et al. [29] reported that the extract of Salvadora persica was effective against Streptococcus mutans and Enterococcus faecalis. In another study, Almas et al. [30] could find that the immediate antimicrobial effects of Salvadora persica high in the case of Streptococcus mutans whereas it was not that effective in the case of Lactobacillus sp. But the present in vitro study shows that the effect of the Salvadora persica miswak extract is almost the same on both S. mutans (8mm inhibition zone) and L. acidophilus (7 mm inhibition zone). Another investigation by Sofrata et al. [31] showed that the Salvadora persica pieces have strong activity against the microorganisms related with the periodontitis and dental caries when the pieces are suspended above the agar plate or embedded in agar.

A study by Al-Bayati et al. [32] on the effect of Salvadora persica aqueous and ethanol extracts on the seven dental pathogens viz. Staphylococcus aureus, Streptococcus pyogenes, Streptococcus mutans, Lactobacillus acidophilus, Enterococcus faecalis, Actinomyces sp., and Candida albicans showed an impressive result in finding that the aqueous extract inhibited the growth of all of the selected pathogens whereas the ethanolic was ineffective against Lactobacillus acidophilus and Actinomyces sp. The aqueous extract showed the most potential activity against Enterococcus faecalis. The antifungal activity was almost equal in the case of both the extracts against Candida albicans when analyzed by turbidity tests [32]. Another study by Chelli-Chentouf et al. [33] on the activity of Salvadora persica extract on selected bacteria showed the highest inhibition against E. coli. In this case, the more potential activity by the ethanolic extract was against Gram-negative (6.5-12 mm) bacteria when compared with the Gram-positive (1–8 mm) bacteria. This could be explained by the variation in the cell wall composition, including the amount of lipopolysaccharides (LPS) layer [34]. Also, this LPS layer makes Gramnegative bacteria comparatively more resistant to antimicrobial compounds. The efficiency of the Salvadora persica ethanolic extract on aerobic bacteria of the oral cavity was also affirmed by Al Sadhan et al. [35]. They could find that Streptococcus mutans was suppressed in a greater extent than the Lactobacillus sp. The phytochemical analysis result of this investigation is also supported by the findings of many other studies. Several other investigations suggested the presence of alkaloid-alvadorine, trimethylamine, chlorides, terpenes, vitamin C, carbohydrates, sulfur, glycosides, large amounts of fluoride and silica, small amounts of tannins, saponins, flavonoids, and sterols. The roots and bark of the Salvadora persica tree are composed of 27% ash; a high ratio of alkaloids, such as salvadorine and trimethyl amine; chlorides and fluorides; moderate concentrations of silica, sulfur, vitamin C; and small quantities of tannins, flavonoids, saponins and sterols [36-39].

The antibacterial activity of Salvadora persica is supposed to be due to the combined activity of a number of compounds present. According to Vahabi et al. [40] it is due to a combined effect of tannins, chlorides, salvadorine, trimetylamine, thiocynate, nitrates and sulphor. The alkaline compound ‘salvadorine’ present in Salvadora persica is thought to have an antimicrobial effect apart from gingival stimulation [37]. Al-Bayati et al. [32] suggest that the antibacterial effect can also be compounds like tannins, alkaloids, saponins and terpenoids. Also, the clinically detectable gingivitis may be reduced by the astringent effect of tannins by their glucosyl transferase activity, thereby reducing plaque and gingivitis [41]. According to Al-Bagieh et al. [42] the high sulfate content of Salvadora persica had an inhibitory effect on the growth of Candida albicans. The low, acidic 5.5 pH of Salvadora persica extract can also disturb the formation of anitrophile flora. According to Guiraud et al. [43] Staphylococcus sp., Enterobacteriaceae and Enterococci were found it difficult to grow in a medium with acidic pH. Nevertheless Streptococcus sp. and Staphylococcus sp. can grow at this pH. In an interesting study, Lemos et al. [44] could understand that the virulence of Streptococcus mutans resides in three significant attributes: tooth surface biofilms farming ability, ability to produce organic acids and the ability to tolerate environmental stresses, including low pH. The promoter effect of components in the extracts of Salvadora persica was hypothesized by Darout et al. [45] which lead to possible antimicrobial activities. Also, eugenol exerts antibacterial, anti-inflammatory and local anaesthetic effects on the dental pulp [46]. Sofrata et al. [47] identified a volatile compound, the isothiocyanate. Root extracts of Salvadora persica as well as commercial synthetic benzyl isothiocyanate exhibited rapid and strong bactericidal effects against oral pathogens involved in periodontal diseases, such as Porphyromonas gingivalis (ATCC 33277) and Aggregatibacter actinomycetemcomitans HK 1519, as well as against other Gram-negative bacteria (Escherichia coli) laboratory strain MC4100 and Escherichia coli K12 LPS-mutant strain D21f2). On the other hand, Gram-positive bacteria, such as Lactobacillus acidophilus (NCTC 1723) and Streptococcus mutans (CCUG 27624) mainly displayed growth inhibition or remained unaffected. Many studies have been carried out on different types of chewing sticks and focused primarily on the antimicrobial activity of those sticks.

Conclusion

Our present study, which analyzed the activity of ethanol extract of Salvadora persica root on selected root canal bacteria finds that Salvadora persica the most widely used chewing stick, has the potential to have an antibacterial effect on root canal bacteria and its repeated use can provide good oral health. Several descriptive studies conducted by various researchers worldwide also confirm the results of the investigation. The detailed studies on phytochemical constituents responsible for this activity of these potential compounds are required. In any case, the Salvadora persica miswak can be recommended for complete oral health care in the form of toothbrush, toothpick, or mouth wash because of its effectiveness, neutrality, availability, simplicity and inexpensive nature.

Acknowledgement

The authors are grateful to the Prince Sattam bin Abdulaziz University, Al-Kharj, Saudi Arabia for its support and encouragement in conducting the research and publishing this report.

Conflict of Interest

Authors declare no conflict of interest.

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