Volume 8, Issue 1 (1-2023)                   J Res Dent Maxillofac Sci 2023, 8(1): 18-27 | Back to browse issues page

Ethics code: IR.IAU.DENTAL.REC.1399.126


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Tavazo Zadeh E, Aref P, Askarizadeh N, Emadi F. In vitro Antimicrobial Effect of Punica granatum Extract versus Chlorhexidine on Streptococcus sobrinus, Streptococcus sanguinis, and Candida albicans. J Res Dent Maxillofac Sci 2023; 8 (1) :18-27
URL: http://jrdms.dentaliau.ac.ir/article-1-395-en.html
1- Faculty of Dentistry, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
2- Department of Pediatric Dentistry, Faculty of Dentistry, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran , parissa234@yahoo.com
3- Department of Pediatric Dentistry, Faculty of Dentistry, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
4- Traditional Persian Medicine, Traditional Medicine Clinical Trial Research Center, Faculty of Medicine, Shahed University, Tehran, Iran
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Abstract

Background and Aim: Considering the search for an effective antimicrobial agent comparable to chlorhexidine (CHX), this study aimed to assess the antimicrobial effect of Punica granatum (P. granatum) hydroalcoholic extract on Streptococcus sobrinus (S. sobrinus), Streptococcus sanguinis (S. sanguinis) and Candida albicans (C. albicans), in comparison with CHX.  
Materials and Methods: In this in vitro study, the disc diffusion test was used to assess the antimicrobial activity of the extract by measuring the growth inhibition zones; while, the microdilution and macrodilution broth tests were applied to find the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extract against the tested microorganisms. The MBC was measured using the blood agar or Mueller Hinton agar culture medium. The Sabouraud dextrose agar culture medium was used for C. albicans. Each test was repeated in triplicate, and data were analyzed by independent samples t-test and Mann-Whitney U test.
Results: None of the tested microorganisms showed any resistance to the extract. CHX had the highest antimicrobial effect against all tested microorganisms. The MIC of the hydroalcoholic extract of P. granatum was 2.5 mg/mL for S. sobrinus and S. sanguinis, and 5 mg/mL for C. albicans. Its MBC was 5 mg/mL for S. sobrinus and S. sanguinis, and 10 mg/mL for C. albicans. The mean diameter of the growth inhibition zone for S. sobrinus caused by CHX was significantly greater than that caused by P. granatum extract (Mann-Whitney U test, P=0.043). The same result was obtained for S. sanguinis (Student sample t-test, P=0.002), and C. albicans (Mann-Whitney U test, P=0.046).
Conclusion: The hydroalcoholic extract of P. granatum has bacteriostatic and bactericidal effects on S. sanguinis and S. sobrinus and antifungal effect on C. albicans comparable to CHX.
Key Words: Pomegranate; Microbial Sensitivity Tests; Antibacterial Agents; Disk Diffusion Antimicrobial Tests; Biological Products


Introduction

Dental clinicians are increasingly interested in using natural organic products for inhibition of dental caries. Effective regular mechanical plaque removal is the basis for almost all preventive and therapeutic approaches for dental caries and chronic periodontal disease. At present, toothbrushing with toothpaste is still the most effective and most practical method to control supragingival plaque in most patients [1,2]. Nonetheless, plaque removal may be ineffective, difficult, or even impossible in children and adults with sensory and/or motor disabilities [3]. If plaque accumulation cannot be prevented, it often leads to the development of dental caries and subsequent destruction of periodontal tissue, bone loss, and eventual tooth loss [4]. Therefore, it is logical to find safe and effective alternatives or adjuncts for plaque control [5,6].
Mouthwashes are among the most effective antimicrobial agents used as an adjunct for plaque control [7]. Chlorhexidine (CHX) has long been the gold-standard antimicrobial mouthwash [8]. Its role in reducing microbial plaque and oral microorganism count and improvement of gingival health status has been previously documented [9-11]. However, it has side effects such as staining, alteration of taste, oral burning sensation, and interference with normal microbial oral flora [12]. Thus, attempts are ongoing to find equally effective alternatives with fewer side effects. Evidence shows that pomegranate fruit or Punica granatum (P. granatum) has antimicrobial activity attributed to its peel, internal white membranes and seeds, and may be able to serve as a natural antimicrobial agent.
Streptococcus sobrinus (S. sobrinus) is a Gram-positive microorganism present in dental plaque, which can cause dental caries and abscess in children [13]. It is the most important cariogenic microorganism responsible for caries development on smooth surfaces [14]. Streptococcus sanguinis (S. sanguinis) is a Gram-positive microorganism that belongs to the family of viridans streptococci and is among the most critical constituents of primary dental plaque [15]. Candida albicans (C. albicans) is another culprit found in the oral cavity and gastrointestinal system of 40-60% of adults. It can serve as a pathogenic microorganism in immunocompromised patients and can cause oral candidiasis. It is also involved in development of dental caries [16].
Studies regarding the antimicrobial activity of the hydroalcoholic extract of the whole pomegranate fruit against S. sobrinus, S.
sanguinis,
and C. albicans are limited
[17-19]. Thus, this study aimed to assess the effect of hydroalcoholic extract of P. granatum on S.
sobrinus, S. sanguinis,
and C. albicans.



Materials and Methods
In this in vitro experimental study, standard-strain S. sobrinus (PTCC 1601), S. sanguinis (PTCC 1449), and C. albicans (ATCC 10231) were obtained from the Iranian Collection of Industrial Microorganisms.
The Ethics Committee of the Faculty of Dentistry at Islamic Azad University provided ethical approval for this study (IR.IAU.DENTAL.REC.1399.126). The sample size was calculated to be 18 according to a previous study
[20-22], assuming 99% confidence interval, 99% study power, beta=0.00, and alpha=0.01 by PASS 11 software; 96-well plates with three repetitions for each concentration of extract were used.

Preparation of hydroalcoholic extract of P. granatum:
Three medium-size pomegranates were purchased, and the entire fruit, including its peel, white membranes, and seeds, were diced into small pieces and crushed in a blender. The water-ethanol solvent in 50:50 ratio was added to the mixture for maceration, and it was filtered with Whatman No.1 filter paper after 24 hours of extraction. The hydroalcoholic extract was then heated at 60°C to concentrate and acquire a honey-like consistency. The concentrated extract was then used to prepare different concentrations [23].
Preparation of microorganisms:
Blood agar, Mueller Hinton agar, and brain heart infusion (BHI) broth were purchased and autoclave-sterilized according to the manufacturers' instructions. The viable bacteria were then cultured on blood agar nutrient under anaerobic conditions in a Gas-Pak jar at 37°C for 24 hours to proliferate and reach maximum confluence. Subculture of all microorganisms was performed three times a week to use fresh microorganisms for each test.
Disc diffusion test:
The disc diffusion test was first performed according to the Clinical & Laboratory Standards Institute recommendations to assess the extract's antibacterial potential [24]. After 24 hours of incubation of microorganisms on blood agar, a certain amount of each microorganism was transferred from the nutrient culture medium to saline to prepare a microbial suspension with 0.5 McFarland standard concentration containing 1.5 x 108 colony forming units (CFUs)/mL. Using a sterile swab, the microbial suspensions were then streak-cultured on Mueller Hinton agar.
A disc containing 0.12% CHX (as the positive control group), a disc containing P. granatum extract (test group), and a blank disc (as negative control) were all placed on the culture medium. The Petri dish was capped and incubated at 37°C for 24 hours. After 24 hours the diameter of the growth inhibition zones was measured by a millimeter ruler. Values < 1 mm were considered zero. To ensure accuracy of the results, each test was repeated under aseptic conditions three times. Each material's antibacterial activity was determined by measuring the diameter of growth inhibition zone around each disc and reported in millimeters (mm)
[25].

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the extract:
The microdilution and macrodilution broth tests, recommended by the Clinical & Laboratory Standards Institute, were performed to determine the MIC and MBC of the extract using BHI broth. For this purpose, a certain amount of bacteria was transferred from the Mueller Hinton agar to saline to obtain a microbial suspension with 0.5 McFarland standard concentration containing 1.5 x 108 CFUs/mL.  To determine the MIC by the microdilution broth test, a 96-well plate was used; 100 µL of the BHI broth was added to all wells by a multi-channel sampler. Next, 100 µL of the pure extract was added to the first well, and after optimal mixing, 100 µL of the contents of this well was transferred to the second well, and so on, until the 10th well.
Finally, 100 µL of the contents of the 10th well was discarded. Next, 100 µL of saline  containing S. sobrinus, S. sanguinis, or C. albicans (0.5 McFarland standard concentration) was added to the wells. The same was performed for 0.12% CHX (positive control).

To determine the MIC by the macrodilution test, the abovementioned steps were repeated but with a larger volume (> 1 cc) in mini-tubes along with the positive and negative control groups. The positive control tube contained BHI broth and the microbial suspension (which had to become turbid); while, the negative control tube only contained BHI broth (which had to remain clear). Next, both microplates and macroplates were placed in an incubator and incubated at 37°C for 24 hours under anaerobic conditions. The turbidity of the tubes was visually evaluated to determine the MIC in milligrams per milliliter (mg/mL). In each series of tests, some tubes remained clear after 24 hours of incubation, indicating no bacterial growth in presence of the extract's respective concentration. The first well/tube that remained clear indicated the MIC of the extract.
To determine the MBC, samples of three macro-tubes, irrespective of their turbidity or clarity, were streak-cultured on blood agar or Mueller Hinton agar culture medium with 5% sheep blood (for streptococci) or Sabouraud dextrose agar (for C. albicans). After 24 hours of incubation at 37°C under anaerobic conditions, the first Petri dish with no bacterial or fungal growth indicated the MBC and minimum fungicidal concentration (MFC) of the extract [26-29].
The Shapiro-Wilk test was applied to assess the normal distribution of data. The independent samples t-test and ANOVA were used to analyze the normally distributed data; while, the Mann-Whitney U and Kruskal-Wallis tests were applied for non-normally distributed data. All statistical analyses were performed using SPSS version 25 at 0.05 level of significance.

Results

 Table 1 presents the MIC and MBC of P. granatum extract and CHX against the microorganisms. Table 2 presents the mean diameter of the growth inhibition zones. As shown, CHX created the maximum growth inhibition zone for all microorganisms.
After CHX, P. granatum extract created the maximum growth inhibition zone in S.
sanguinis
culture.

Table 3 and Figure 1compare the diameter of the growth inhibition zones of all three microorganisms around the P. granatum extract and CHX discs.  As shown, the mean diameter of the growth inhibition zone of S. sobrinus caused by CHX was significantly greater than that caused by P. granatum extract (non-normal distribution, Mann-Whitney U test, P=0.043). The same result was obtained for S. sanguinis (normal distribution of data, Student sample t-test, P=0.002) and C. albicans (non-normal distribution, Mann-Whitney U test, P=0.046).

Table 1. MIC and MBC of P. granatum extract and CHX against the microorganism
Table 2. Mean diameter of growth inhibition zone
Table 3. Comparison of the diameter of growth inhibition zones of all three microorganisms around the P. granatum extract and CHX discs
Figure 1. Comparison of the diameter of growth inhibition zones of all three microorganisms around the P. granatum extract and CHX discs


Discussion

This study assessed the effect of 50%-50% hydroalcoholic extract of P. granatum on S. sobrinus, S. sanguinis, and C. albicans compared with 0.12% CHX. The MIC and MBC of the extract against all three microorganisms were also determined. The results revealed that none of the tested microorganisms showed any resistance to the extract. CHX had a significantly higher antimicrobial effect than the extract against all tested microorganisms. The MIC of the hydroalcoholic extract of P. granatum was 2.5 mg/mL for S. sobrinus and S. sanguinis, and 5 mg/mL for C. albicans. Its MBC was 5 mg/mL for S. sobrinus and S. sanguinis, and its MFC was 10 mg/mL for C. albicans
Pomegranate is a natural source of phenolic compounds and antioxidants such as tannin, ellagitannin, punicic acid, polyphenol, flavonoids, and vitamin C. It also contains tocopherol and anthocyanins [30]. Tannin-rich ellagitannins and phenolic acids have antibacterial properties, probably due to their secondary metabolites [31]. Phenolic compounds, particularly gallic acid, have significant antibacterial activity [32]. Tannins and polyphenolic compounds are the main phytochemical agents   in pomegranate [33]. Tannins can pass through the cell wall of microorganisms and degrade the membrane proteins, destroying the microorganisms. They also inhibit enzymes such as glycosyltransferase that play a key role in the adhesion of bacteria to tooth surfaces and prevent bacterial adhesion [34]. Tannins have different mechanisms of action against Candida species. They exert their antifungal effect by inhibition of extracellular microbial enzymes and depletion of nutrients and iron. Also, they directly affect microbial metabolism by inhibiting oxidative phosphorylation [35]. Evidence shows that P. granatum extract boosts the activity of several antibiotics such as chloramphenicol, gentamycin, ampicillin, tetracycline, and oxacillin against 30 S. aureus strains [36]. The pomegranate peel extract has maximum antimicrobial activity compared with other parts of this fruit due to the presence of polyphenolic compounds [34,37]. The antibacterial activity of the white membranes and internal parts of the fruit is due to the presence of phytocompounds such as hydrolyzing tannins, polyphenols, and flavonoids, including punicalagin, gallic acid, and catechins [38,39]. The phenolic compounds in pomegranate peel extract can react with microorganisms' cellular proteins, change their cell wall structure, and denature some microbial enzymes.
Also, phenolic compounds form complexes with carbohydrates, proteins, vitamins, and minerals and cause their depletion. By doing so, they adversely affect the metalloenzymes, degrade the cell wall, and prevent coaggregation of microorganisms [40]. Polyphenols also significantly decrease the bacterial load by reducing the environmental pH [41].
Evidence shows that punicalagin is the most effective antibacterial compound in a pomegranate; however, the pomegranate fruit's synergistic effects are mainly responsible for its cariostatic properties
[42].

Evidence shows that ethanolic extract (65-85%) maintains the tannins, polyphenols, and procyanidines and has inhibitory effects on S. aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa (P. aeruginosa), and C. albicans [43]. Also, it has been reported that the whole fruit extract of pomegranate has higher antioxidant activity than the extract of each component alone [44]. Thus, the whole fruit extract was evaluated in this study. Gosset-Erard et al. [39] identified punicalagin as the main antibacterial agent present in pomegranate peel. Among the evaluated ex-tracts in their study, ethanolic extract had the greatest antimicrobial activity with a growth inhibition diameter of 20 mm, which was close to the present findings. This effect was attributed to active antimicrobial agents in pomegranate peel. The MIC of a and b forms of punicalagin was 0.3 and 11.2 µg/mL, respectively. Veloso et al. [45] evaluated the antibacterial activity of medicinal herbs including pomegranate against oral bacteria. The diameter of growth inhibition zones ranged from 1.6 to 10.3 mm. Pomegranate ethanolic (80%) extract was the only extract that inhibited all the tested microorganisms and had high level of polyphenols. It had a MIC of 50 to 400 µg/mL. Their results were in agreement with the present findings. Ferrazzano et al. [46] confirmed the optimal antibacterial activity of hydroalcoholic extract of pomegranate peel and pomegranate juice against cariogenic bacteria. They reported that pomegranate peel extract with MIC and MBC of 10 and 15 µg/µL, respectively, inhibited the proliferation of cariogenic bacteria; while pomegranate juice had inhibitory effects on Streptococcus mutans (S. mutans) with MIC and MBC of 25 and 40 µg/µL, respectively. The concentration of polyphenols in the pomegranate peel is higher than that in the pomegranate juice. Thus, the peel extract has higher antimicrobial activity than the pomegranate juice. The MIC and MBC values reported in their study were higher than those in our study, which may be due to the fact that we used the whole fruit extract, which is more potent. Although they did not measure the growth inhibition zones, the optimal efficacy of the hydroalcoholic extract of P. granatum in their study was in line with our findings. Nozohour et al. [47] evaluated the antibacterial activity of alcoholic extract of pomegranate seed and peel against S. aureus and P. aeruginosa and reported that ethanolic extract of pomegranate had a greater inhibitory effect than tetracycline and chloramphenicol against the above mentioned bacteria. The MIC and MBC of the pomegranate seed extract (25 and 50 mg/mL, respectively) were significantly higher than those of the pomegranate peel extract (12.5 and 25 mg/mL, respectively). However, this difference was not significant for P. aeruginosa. The diameter of the growth inhibition zone caused by the pomegranate peel extract ranged from 22.5 to 27.5 mm (9 mg/disc) while this range was 18.7 to 22 mm (9 mg/disc) for the pomegranate seed extract; these values were close to the values obtained in the present study (11.17 to 15.83 mm). Such results indicate higher antibacterial activity of pomegranate peel extract than seed extract. Their results were in agreement with our findings. Benslimane et al. [31] evaluated the antioxidant and anti-biofilm activity of pomegranate peel extract against the bacteria isolated from carious lesions and subgingival plaque. They prepared the pomegranate peel extract by using 70% acetone, 70% ethanol, and 70% methanol. All tested bacteria were sensitive to the extracts with a MIC of 0.0125 to 100 mg/mL. Gram-positive bacteria were more sensitive. The ethanolic extract showed higher antimicrobial activity. Their results were in line with ours. Millo et al. [48] evaluated the antibacterial effect of a formulated gel of pomegranate fruit against S. mutans, S. sanguinis, and Lactobacillus casei. They performed high-performance liquid chromatography to identify and quantify punicalagin and determine its MBC. They showed that the formulated pomegranate gel had an optimal inhibitory effect on S. mutans, although its effect was smaller than that of CHX. They also reported that the amount of punicalagin was 2033.58±25.29 pg/mL in aqueous extract of P. granatum and 0.234 pg/mL (w/w) in the formulated gel. The MBC of the extract was 500, 250, and 125 mg/mL for Lactobacillus casei, S. mutans, and S. sanguinis, respectively. The antibacterial activity of the formulated gel in their study was lower than the hydroalcoholic extract activity in our study, which can be due to the lower amount of punicalagin in the formulated gel.
Aravindraj et al.
[49] evaluated the antimicrobial effect of ethanolic, methanolic, acetone, ethyl acetate, chloroform, hexane, and dimethyl sulfide extracts of pomegranate against S. aureus, S. mutans, Lactobacillus acidophilus, Enterococcus faecalis, and C. albicans. They reported a significant difference between different extracts. They reported a 9-12 mm diameter of growth inhibition zone for C. albicans, close to the value in the present study (11.17 mm). Anibal et al. [17] evaluated the antifungal activity of 70% ethanolic extract of aril, seed, pericarp, peel, and the whole fruit of pomegranate and the resultant morphological changes in the structure of C. albicans. The pure ethanolic extract was analyzed by mass spectrometry, and a number of ingredients such as punicalagin were isolated. The pericarp and peel extracts with 125 µg/mL MIC had inhibitory effects on C. albicans. The effect of pericarp and seed extract on the morphology and structure of C. albicans was evaluated by transmission electron microscopy. The results confirmed the antimicrobial potential of ethanolic extract of P. granatum against Candida genus and the ability of its bioactive compound to change this microorganism’s structure and morphology. The obtained MIC value was lower than the value in our study, which can be due to the use of pure ethanolic extract in their study. However, their results were generally in agreement with our findings.

This study did not separately assess the antimicrobial activity of the constituents of the extract. Thus, future studies are required to assess the antimicrobial activity of the isolated fractions of pomegranate extract. Also, P. granatum extract's antibacterial activity should be investigated against a broader spectrum of pathogenic microorganisms, particularly Gram-negative bacteria. Moreover, the cytotoxicity of P. granatum extract against human oral fibroblasts should be evaluated in vitro. Given that its safety is confirmed, clinical trials can be carried out to assess its use in the clinical setting and its mass production in the form of mouthwash, chewing gums, or lollypops.

Conclusion

The hydroalcoholic extract of P. granatum has bacteriostatic and bactericidal effects on S. sanguinis and S. sobrinus and antifungal effect on C. albicans.


Type of Study: Original article | Subject: Restorative Dentistry

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