Tribulus terrestris (TT) extract has shown good antibacterial activity against some bacteria. However, there are limited data on its cariogenic properties. This in vitro study aimed to evaluate the antibacterial and antibiofilm activities of TT extract against Streptococcus mutans (S. mutans), Streptococcus sobrinus (S. sorbinus), and Lactobacillus acidophilus (L. acidophilus) as the important cariogenic bacteria.
Materials and Methods:
This study was designed in an experimental model (in vitro). Phytochemical tests were carried out to detect herbal compounds in the TT extract. Agar well diffusion was performed to compare the extract (500–62.5 mg/mL) with different concentrations of chlorhexidine (2–0.25 mg/mL). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the TT extract and chlorhexidine were also determined. The lowest concentration showing ≥50% inhibition of biofilm formation (MBIC50) was determined using crystal violet assay. Further, the time-kill assay (Log of CFU/mL) was performed, and acid production (pH) was measured at 1 × MIC concentration in 2, 4, 8, 12, and 24 h. Data analysis conducted using SPSS software (v26, IBM) involved One-way analysis of variance, Tukey post hoc tests, and t-test to compare concentrations and groups. Significance level is set at 0.05.
Results:
The TT extract mostly consisted of flavonoids. Its inhibition zones in the well diffusion test were statistically comparable with chlorhexidine in some concentrations (P > 0.05). The MIC of the TT extract was 15.625 mg/mL for all tested bacteria, whereas the MBC ranged from 31.25 to 62.5 mg/mL. Further, the MBIC50 ranged from 7.8125 to 15.625 mg/mL for the extract. Time-kill assay showed that the bactericidal activity of the TT extract lasted for 8, 12, and 2 h for S. mutans, S. sobrinus, and L. acidophilus, respectively. The acid production decreased obviously after 8 h.
Conclusion:
The TT extract showed good time-dependent antibacterial and antibiofilm activity, as well as acid production inhibition, against cariogenic bacteria in laboratory experiments.
Dental caries (tooth decay) is known as the most common chronic and infectious disease of the mouth in the world.[1] The mouth provides a unique environment for bacteria to accumulate on tooth surfaces and form the oral biofilm. Cariogenic bacteria produce acid by fermenting carbohydrates, which destroys the surface of tooth enamel.[2,3] Among dental plaque bacteria, Streptococcus spp. plays an important role in the initial phase of dental caries, whereas lactobacilli are very active in the development of tooth decay.[4] Of the 300 species living in dental plaque, Streptococcus mutans (S. mutans) and Lactobacillus acidophilus (L. acidophilus) are the two leading causes of tooth decay.[5] Epidemiological and laboratory studies have shown that Streptococcus sobrinus, another cause of caries, can be even more cariogenic than S. mutans. Clinical studies have also revealed that the co-existence of both S. mutans and S. sobrinus in preschool and 15-year-old children can increase dental caries rates compared to S. mutans alone.[6-8]
To prevent dental caries, a mechanical and chemical approach should be considered to remove and control plaque. Various synthetic chemicals, such as chlorhexidine, are used to control plaque. Despite the sound antibacterial effects of such substances, a variety of side effects, such as discoloration of teeth and restorations, taste disturbance, and desquamation of the oral lining, can be seen in long-term use.[9] Therefore, the search for alternative substances in plants as a possible source of the desired compounds has been considered more than ever.[10,11]
Tribulus terrestris (TT) is an herb belonging to the genus Tribulus and Zygophyllaceae family. It is a perennial flowering plant commonly found worldwide but primarily in warm and tropical areas. TT can also grow in poor climates and soils unsuitable for other herbs.[12] It contains flavonoids, steroids, saponins, alkaloids, unsaturated fatty acids, vitamins, tannins, resins, potassium, nitrate, phosphorus, iron, sodium, calcium, sulfur, chlorine, aspartic acid, and 14-aspartic acid. Furthermore, it has antimicrobial, antioxidant, cyclooxygenase inhibition, free radical scavenging, lipid peroxidation inhibition, and inflammatory agent modulation properties. In traditional medicine, various diseases, such as hypotension, diabetes, renal, cardiovascular, gastrointestinal, sexual (in men), and hepatic disorders, have been treated. This plant is used in traditional medicine in China, India, Iraq, Bulgaria, South Africa, and Iran.[13] Some in vitro studies have shown good antibacterial efficacy of TT extracts against pathogenic and non-pathogenic microorganisms such as Staphylococcus epidermidis, Entrococcus faecalis, Salmonella parathyphi, Shigella dysenteriae, Candida albicans, and Mycobacterium tuberculosis.[14,15] However, only a few researchers have explained its antibacterial effects on cariogenic bacteria.[16,17]
This in vitro study aimed to assess the antibacterial and antibiofilm activities of TT methanolic extract against S. mutans, S. sobrinus, and L. acidophilus as three important etiologic elements of dental caries.
MATERIALS AND METHODS
This study was designed in an experimental model (in vitro), and it was ethically approved by the Research Ethics Committees of Rafsanjan University of Medical Sciences with an ethical code of (IR.RUMS.REC.1400.026).
Plant and bacterial strains
TT fruits were obtained from Pakan Bazr Isfahan Corporation (collected from the Chadegan region of Isfahan province). The standard bacterial strains were also purchased from the Iranian Research Organization for Science and Technology, including S. mutans (ATCC 35668), S. sobrinus (ATCC 27607), and L. acidophilus (ATCC 4356).
Preparation of Tribulus terrestris extract
Plant extraction was performed as described previously with some modifications.[18] Fruits of TT were washed with distilled water and dried in a dark place for 3 days at 20°C. They were completely crushed by an electrical grinder. Two hundred grams of the produced powder were soaked with 1 L of pure methanol (Merck, Germany) for 4 days. After filtering with Whatman paper No. 1, the solvent was removed using a rotary evaporator (Heidolphlaborota 4000, Germany). The dry remaining mass was dissolved in 10% dimethylsulfoxide (DMSO, Merk), resulting in a final concentration of 500 mg/mL. This stock solution was stored at 4°C for the following use.
Phytochemical analysis
Standard preliminary phytochemical tests were performed to identify specific compounds in the TT extract. Mayer’s test, Foam test, Ammonia test, Braymer’s test, Ferric chloride test, Libermann-Burchard’s test, and Salkowski’s test were performed to detect alkaloids, saponins, flavonoids, tannins, phenols, steroids, and terpenoids, respectively.[19]
Antibacterial assaysAgar well diffusion
The inhibitory effect of the TT extract on S. mutans, S. sobrinus, and L. acidophilus was determined by the agar well diffusion assay.[20] Briefly, the fresh bacteria from a 0.5 McFarland suspension (1.5 × 107 CFU/mL) were subcultured on a Mueller–Hinton agar by a swab. The 8 mm wells were created on the mediums, and different concentrations (500, 250, 125, and 62.5 mg/mL) of the TT extract were injected into the wells. All cultures were incubated at 37°C for 24 h. The inhibition zones were measured with a ruler. Furthermore, a diluted series of chlorhexidine (Donya Behdasht, Tehran, Iran) with concentrations from 2 to 0.25 mg/ml was used for comparison. All test was carried out in triplicate for each bacterium and concentration (3 samples for the control versus 3 for the test group), and average zones were reported.
Determination of minimum inhibitory concentrations
The microdilution method was used for minimum inhibitory concentration (MIC) determination. Two-fold dilutions of the TT extract (0.49–250 mg/mL) and chlorhexidine (0.062–2 mg/mL) were prepared in a 96-well microplate. Bacterial suspensions with a concentration of 106 CFU/mL were prepared in BHI broth. 100 μl of this bacterial suspension was added to each well containing 100 μl of the TT extract. The microplate was incubated for 24 h at 37 °C. The concentration with no visible bacterial growth after 24 h of incubation was considered the MIC.[21] These experiments were performed in triplicate (three experiments for the TT extract group and three for the chlorhexidine group) for each bacterium.
Determination of minimal bactericidal concentration
After determining MIC values, 10 μL from each well of the microplate was spread on the BHI agar and incubated for 24 h in a microaerophilic condition. The lowest concentration of the TT extract, killing 99.99% of bacteria, was defined as minimum bactericidal concentration (MBC).[21] This procedure was also conducted in triplicate (three experiments for the TT extract group and three for the chlorhexidine group) for each bacterium.
Time-kill assay
A time-kill kinetic assay was conducted using broth microdilution. The following BHI mediums, including 106 CFU/mL bacteria plus TT extract (1 × MIC) and 106 CFU/mL bacteria plus PBS (negative control), were prepared and incubated at 37°C. After 2, 4, 8, 12, and 24 h of incubation, 100 μl of each bacterial solution was serially diluted with phosphate-buffered saline (PBS, pH: 7). Finally, 10 μl of these dilutions were cultured onto BHI agar for colony counting. Enumeration of CFU was performed after incubation of cultures at 37°C for 24 h in microaerophilic conditions. Time-kill curves were traced with the log10 CFU/mL over the 24 h. This test was performed in triplicate for each bacterium and group (control and test, with 18 time-kill assays in total).
Acid production
Acid production was measured using Matsumoto et al.’s methodology with some modifications.[22] In brief, the TT extract (6.45 mL of 500 mg/mL stock solution) was added to 200 mL of BHI broth (Difco, USA) containing 106 CFU/mL supplemented with 1% glucose, resulting in MIC (15.625 mg/mL) in each broth medium. 5 mL of the cultures were removed at 2, 4, 8, 12, and 24 h after incubation at 37°C, and the pH was measured using a pH meter (Model 220, Corning). A BHI broth without the TT extract was also used as the control. This experiment was performed once for each bacterium and group (control and test) for 24 h, with 6 acid production assays in total.
Biofilm formation assay
The effect of the TT extract and chlorhexidine on biofilm formation was evaluated by the microdilution method. Two-fold serial dilutions of the extract (ranging from 0.49 mg/mL to 250 mg/mL) and chlorhexidine (0.0625–2 mg/mL) were prepared. Bacterial suspensions were prepared in the same manner as in the MIC determination, 20 μL of bacterial suspensions were added in the wells of a 96-well microplate, leading to the final concentration of 1 × 106 CFU/mL. Following incubation at 37°C for 24 h, wells were washed with PBS and fixed with methanol for 15 min. 0.1% (w/v) crystal violet was used for staining in 5 min. Then, all wells were rinsed thoroughly with water, and the control wells became colorless. 200 μL of 95% ethanol was added to each well, shaken for 30 min, and finally, absorbance was measured at a wavelength of 590 nm by a microplate reader. The percentage of inhibition was calculated by the following formula: (1-[absorbance of the test/absorbance of the control]) ×100. The lowest concentration showing ≥50% inhibition of biofilm formation was defined as the minimum biofilm inhibition concentration (MBIC50). This assay was performed in triplicate (three experiments for the TT extract group and three for the chlorhexidine group) for each bacterium.
Statistical analysis
Data analysis was performed with SPSS software (version 26.0; SPSS Inc., Chicago, IL, USA) using one-way analysis of variance (ANOVA) and Tukey post hoc tests to compare different concentrations and groups. T-test analysis was also performed to compare the decrease of bacteria growth in the TT extract group with the control shown in the time-kill assay. The statistical significance level was set at 0.05.
RESULTSPhytochemical analysis
The standard preliminary photochemical tests showed that the majority of the TT extract consisted of flavonoids, as demonstrated in Table 1. It included tannins, phenols, steroids, and terpenoid compounds. The TT extract was devoid of saponin compounds [Table 1].
Phytochemical analysis of Tribulus terrestris
Antibacterial assaysAgar well diffusion
One-way ANOVA showed a statistically significant difference between groups of S. mutans (F [7,16] = 451.95, P < 0.001), S. sobrinus (F [7,16] = 347.54, P < 0.001), and L. acidophilus (F [7,16] = 35.47, P < 0.001). The Tukey post hoc test showed that inhibition zones of 125 and 250 mg/mL of the TT extract against S. mutans did not significantly differ from 0.5 and 0.25 mg/mL of chlorhexidine, respectively. The data showed no significant difference between 500 and 250 mg/mL of the TT extract with 0.5 mg/mL of chlorhexidine on S. sobrinus. Similar results were also found between 125 mg/mL of the TT extract and 0.25 mg/mL of chlorhexidine (P > 0.05).
The inhibition zone of 250 mg/mL of the TT extract against L. acidophilus was not significantly larger than 0.25 and 0.5 mg/mL of chlorhexidine, according to the Tukey post hoc test. Moreover, 500 mg/mL and 62.5 mg/mL of the extract had statistically similar zones to chlorhexidine with concentrations of 0.5 and 0.25 mg/mL, respectively (P > 0.05). The comparison results between all other concentrations of the TT extract and chlorhexidine were statistically significant, in which the agent showing a larger inhibition zone was statistically more effective (P < 0.05). The results of the agar well diffusion assay are presented in Table 2.
Antimicrobial activities of the Tribulus terrestris extract and chlorhexidine against tested microorganisms in millimeters using the well diffusion method (mean±standard deviation) (n=3; for each concentration and bacteria)
Minimum inhibitory concentration, minimum bactericidal concentration, and MBIC<sub>50</sub>
MIC, MBC, and MBIC50 values of the TT extract and chlorhexidine are reported in Table 3.
Minimum inhibitory concentration, minimum bactericidal concentration, and minimum biofilm inhibition concentration 50 values of the Tribulus terrestris extract and chlorhexidine against tested microorganisms (n=3; for each test and bacteria)
Time-kill assay
According to the t-test analysis of the time-kill assay, the TT extract had a significant ability to inhibit the growth of all three tested bacteria in all periods in comparison with control groups (P < 0.05) except for L. acidophilus in 24 h (P = 0.371). The bactericidal activity of the TT extract lasted 8 h, 12 h, and 2 h for S. mutans, S. sobrinus, and L. acidophilus, respectively, showing no visible viable colonies. All test bacteria showed visible growth after the mentioned period [Figure 1]. L. acidophilus was the most resistant bacteria, whereas S. sobrinus was the most susceptible with longer growth inhibition. Details of Log CFU/mL of per bacteria strains are presented in Table 4.
Time-kill assay of bacteria strains during 24-h exposure to Tribulus terrestris extract (1 × minimum inhibitory concentration) compared to negative control; (a) Streptococcus mutans, (b) Streptococcus sobrinus, (c) Lactobacillus acidophilus (n = 3 for each group).
Log CFU/mL of bacteria strains during 24-h exposure to Tribulus terrestris extract (1× minimum inhibitory concentration) in comparison with negative control in triplicate
Acid production
Acid production of all tested microorganisms did not decrease compared with negative control up to 4 h. After 8 h, a decrease in acid production rate was evident in the three bacteria [Figure 2]. Details of pH changes are provided in Table 5.
pH changes during 24-h exposure of bacteria strains to Tribulus terrestris extract (1 × minimum inhibitory concentration) compared to negative control; (a) Streptococcus mutans, (b) Streptococcus sobrinus, (c) Lactobacillus acidophilus (n = 1; for each bacterium and group).
pH changes during 24-h exposure of bacteria strains to Tribulus terrestris extract (1× minimum inhibitory concentration) in comparison with negative control
DISCUSSION
The TT extract has been widely used for the treatment of toothache, dental caries, and periodontal diseases in traditional Chinese medicine.[23,24] Three-time oral rinse with the TT extract has also been recommended for the healing of gingivitis in the traditional herbal medicine of Mexico.[25] Another in vitro study has also strengthened the theory of this extract’s usefulness in oral and dental health.[17]
In the present study, the results of phytochemical screening of the methanolic extract of the native TT in the Chadegan region of Isfahan demonstrated that the extract contained flavonoids (the main compound), alkaloids, tannins, phenols, steroids, and terpenoids. Oh et al. showed that the ethanolic TT extract had a wide range of constituents, including phenols, glycosides, peptides, flavonoids, steroids, terpenoids, and organic acids. No alkaloids were detected, whereas steroids and terpenoids were the maximum secondary metabolites.[16]T. terrestris, which grows in Punjab in Pakistan, had no tannins, whereas other compounds, such as terpenoids, phlorotannins, anthraquinone, and phenolics, were present as the majority of flavonoids.[18] It has been revealed that different constituents of the TT extract are linked to different factors, such as plant origin, climate conditions, and geographical region. Moreover, the extraction method and duration can also affect the final composition of the extract.[26]
The TT extract could significantly inhibit three bacteria in comparison with previous studies. In Soleimanpour et al., well diffusion tests showed that 50 and 100 mg/mL of the TT extract led to zones of 21.8 ± 0.3 and 22.2 ± 0.2 mm against S. mutans, respectively. Zones of 18 ± 0.0 and 20.4 ± 0.5 mm were also reported against S. sanguis for 50 and 100 mg/mL, respectively.[17] Moreover, the methanolic extract of TT fruits showed good antibacterial activity against Staphylococcus aureus, Klebsiella pneumonia, and Acinetobacter baumannii.[18] Inhibition zones of chlorhexidine against S. mutans and S. sanguis were 18 ± 0.0 and 14.2 ± 0.2 for 0.5 mg/mL and 15 ± 0.0 and 13.7 ± 0.99 mm for 0.25 mg/mL, respectively.[17] Different bacteria species, extraction methods, and plant origins can cause various inhibition zones.[26] To our knowledge, limited studies have compared the TT extract with chlorhexidine, especially against S. sobrinus and L. acidophilus. The present study suggested that the antibacterial activity of the TT extract could not significantly differ from chlorhexidine in some concentrations. Furthermore, this could be the high concentration of the TT extract or its richness in various medicinal compounds according to the phytochemical screening results.
MIC and MBC of the TT extract have been studied previously. MIC and MBC of the aqueous extract of TT against S. mutans and S. sanguis were higher than the reported concentration for methanolic extract in the present research. On the other hand, the MBC value reported by Kiani et al. was lower than that in our study; however, it was higher than the MBC value reported by Soleimanpour et al. against S. mutans.[17,27] The TT methanolic extract seems more effective than the aqueous extract, suggesting a higher concentration of antibacterial substances released from the methanolic extract.
According to our results, biofilm formation can be significantly inhibited in exposure to the TT methanolic extract. The ethanolic extract of TT fruits can inhibit the adhesion of S. mutans to saliva-coated hydroxyapatite and the synthesis of water-insoluble glucan in a dose-dependent manner (0.1–0.5 mg/mL).[16] The cell surface hydrophobicity of S. mutans has an important role in biofilm formation. It has been shown that other strains, such as S. sanguis and S. salivarius, are not able to adhere to hydroxyapatite due to the lack of this property.[28,29] Further studies are needed to find whether the TT extract can reduce the cell surface hydrophobicity. Studies have demonstrated that the existence of strongly attached and insoluble glucans within a biofilm enhances its mechanical stability by binding bacterial cells together and the apatite surface. These polymers are crucial for maintaining the three-dimensional structure of the biofilm, in addition to interacting with the proteins related to the specific Gbps genes expressed by S. mutans and other oral microorganisms. As a result, they play a role in regulating the development of cariogenic biofilm.[30-32] This may explain the biofilm formation reduction of other Streptococci spp., whereas the main mechanism for lactobacilli remains unclear.
The time-kill assay results of the present research showed the bactericidal effect of the TT extract to be time-dependent for S. mutans, S. sobrinus, and L. acidophilus. Tolerance and resistance to herbal drugs have also been reported for Streptococci and Lactobacilli spp.[33] This can be caused by nutrient compounds in the TT extract or the creation and growth of resistant strains through time. This new data are hard to discuss due to the absence of a similar study.
Based on the present study, the TT extract can reduce acid production after 8 h compared to the control. These results are in line with Oh et al.’s findings after 24 h, suggesting that methanolic extract can inhibit the acid production of S. mutans.[16] The other two bacteria examined in our study have been investigated for the first time. Oh et al. suggested that the anticariogenic properties of the TT extract against S. mutans, including reducing acid production, could be related to active compounds of TT.[16]
The main components responsible for the antibacterial and antibiofilm effects of the TT extract are unclear. Some studies have mentioned alkaloids,[34] whereas some have mentioned saponins as the main compound.[35,36] Flavonoids of the TT extract have also been proven to have antibacterial activity against different species, such as Streptococci.[37,38] The TT extract of the present study also showed high amounts of flavonoids, which can explain the antibacterial and antibiofilm effects in line with the previous studies.[37,38]
CONCLUSION
The TT methanolic extract seems to have good time-dependent antibacterial activity against cariogenic bacteria. It could inhibit the growth, biofilm formation, and acid production of S. mutans, S. sobrinus, and L. acidophilus. However, in vivo studies are needed to find out whether the TT extract or its compounds are a possible candidate for use in oral health products.
Financial support and sponsorship
Nil.
Conflicts of interest
The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.
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