The effect of pomegranate peel extract and apple cider vinegar as an irrigants in disinfecting the necrotic primary root canal

Abstract

This in-vivo study aims to assess pomegranate peel extract (PPE) and Apple Cider Vinegar (ACV) efficacy in disinfecting the necrotic primary teeth root canals. Forty-five primary incisors in thirty children. The treatment was performed under local anesthesia with rubber dam isolation. Sterile paper points were inserted for 60 s to collect the first bacterial swab (S1). Root canal walls were prepared and irrigated with 22 mL within 5 min according to the following groups (randomly with 1:1:1 allocation ratio): Group A: (n = 15) with 5% PPE, Group B: (n = 15) with 5% ACV, and Group C: (n = 15) with 5% sodium hypochlorite (NaOCl). Canals were dried with paper points, and paper point was placed in each canal for 60 s to collect the second bacterial swab (S2). Bacterial colonies were counted after 48-hour incubation at 37 °C and the bacterial counts in the S1 and S2 for each sample were compared using the decimal logarithm. Intra-group comparisons were made using Paired sample t and Wilcoxon signed ranks tests. Between-groups comparisons were made using One-Way ANOVA, Kruskal-Wallis, Mann-Whitney U, and Independent Samples tests. The significance level was set at α = 0.05. PPE demonstrated a 60.43% reduction in CFU count, ACV demonstrated a 51.56% reduction in CFU count, while NaOCl showed an 87.49% reduction in CFU count. Although NaOCl has shown effectiveness in reducing bacterial counts, PPE and ACV have also displayed some antibacterial activity. However, their overall efficacy is less established, which raises questions about their viability as a natural alternative to a 5% NaOCl.

Introduction

A primary objective of endodontic treatment is to diminish the overall microorganisms and bacteria within the root canal system, thereby ensuring effective disinfection. This involves focusing on mechanical preparation, irrigation, and proper root canal obturation¹.

Relying on mechanical preparation alone is unlikely to achieve such disinfection, considering factors such as root resorption, the intricate root canal system anatomy, and the potential risk of harming the permanent tooth buds².

Presently, irrigation is recognized as the most efficient technique for disinfecting root canal systems by eradicating bacteria, removing the smear layer, and eliminating any remnants from the preparation and shaping procedures³. In clinical practice, various intracanal irrigants have been suggested for primary teeth, including sodium hypochlorite (NaOCl), chlorhexidine (CHX), and hydrogen peroxide³. While NaOCl is a commonly used canal irrigant in endodontic treatments³, some dental professionals advise against its use in primary teeth due to potential complications after application such as tissue-related toxicity, the potential for allergic reactions, an unpleasant taste, and limitations in completely removing the smear layer^4–7. Moreover, elevated concentrations application of NaOCl can lead to unfavourable consequences, including toxic reactions from excessive fluid overflow, detrimental impacts on dentin flexibility and durability, decreased dentin microhardness, and possible harm to stem cells in the apical region⁸.

This issue is especially critical in primary teeth, as the irrigant flow through the apical zone, driven by natural resorption processes, may cause harm to the succeeding permanent teeth⁹.

Various herbal or plant-based products are gaining popularity due to their enhanced antimicrobial properties, biocompatibility, anti-inflammatory, and antioxidative characteristics¹⁰. Over time, various traditional herbal-based remedies have been employed in medical traditions. There is a growing emphasis on integrating these natural plant products into dental and oral health issues management¹¹.

Punica granatum (pomegranate) is native to the region from northern India to Iran. It is also widely cultivated in Southwest America, California, Mexico, Arizona and Africa^12,13.

Studies have shown that pomegranate has a wide range of potential effects, including bacteriocidal, antifungal, antiviral, immune modulation, vermifuge, stimulant, refrigerant, astringent, stomachic, styptic, laxative, diuretic, and anthelmintic properties. Additionally, it has been found to alleviate the adverse effects of various conditions such as bruises, sores, mouth lesions, skin lesions, periodontal diseases, and denture stomatitis, the anti-inflammatory, antioxidant, and antimicrobial characteristics of pomegranate could be advantageous in the aphthous ulcer’s treatment^12–17.

An in-vitro study demonstrated that the combination of pomegranate and CHX exhibited greater efficacy against E. faecalis compared to the combination of pomegranate and NaOCl¹⁸. Furthermore, the effect of pomegranate peel extract (PPE) as an irrigant for root canals in primary teeth has been studied compared to chitosan and EDTA. The results indicated that PPE had the least effect on the hardness of dentin¹⁹.

Apple cider vinegar (ACV) has found applications in various medical fields. It demonstrates antibacterial properties, helps in diabetes and cardiovascular disease management, and has effects in treating conditions such as nail fungus, head lice, warts, and ear infections²⁰.

Research into the precise mechanism of acetic acid’s effects on cells reveals that the organic acids present in vinegar, particularly acetic acid, penetrate the cell membranes of microorganisms, resulting in their demise²⁰. Moreover, ACV has the same effect as 5% NaOCl on faecal enteric bacteria but is less effective than NaOCl against C. albicans²¹.

Therefore, this study aimed to determine the effect of 5% ACV and 5% PPE compared to 5% NaOCl in reducing bacterial counts within the root canals of necrotic anterior primary teeth.

The null hypothesis tested was that PPE and ACV are equally effective as 5% NaOCl in reducing bacterial counts within root canals, with no significant differences.

Materials and methods

Study design, settings, and ethical approval

This study design was a parallel arm, double blinded, randomized, clinical trial with an active control group. The study was approved by the Biomedical Research Ethics Board at the Damascus University (Identification number: DN-241023), and performed in accordance with the Helsinki Declaration. The study was conducted at the Department of Pediatric Dentistry, Faculty of Dentistry, Damascus University. The trial was registered in the ISRCTN database, with the following ID: ISRCTN79064400 in in 02/06/2023. The CONSORT statement for reporting randomized controlled trials was applied in this clinical part of the research. The study took place between June 2023 and April 2024. Parents of enrolled children have signed an informed consent before the study initiation.

Sample size calculation

The sample size in the present study was calculated by G* Power 3.1.9.4 (Heinrich-Heine-Universität, Düsseldorf, Germany) based on the mean logarithm of bacterial reduction changing. A minimum total sample size of 45 subjects (15 in each group) was found to be sufficient for a level of significance of 0.05, a power of 85%, and an effect size d of 1.05 that estimated from a previous study¹⁴.

Randomization

The randomization process involved the use of cards detailing the irrigant of the treatment (PPE, ACV, or NaOCl), which were enclosed in opaque, sealed envelopes and then randomly numbered using an Excel spreadsheet’s random allocation function (Microsoft, Washington, USA). Patients were directed to select an envelope at random, leading to their assignment into one of three groups: Group 1, comprising 15 teeth irrigated with 5% PPE, Group 2, comprising 15 teeth irrigated with 5% ACV, and Group 3 comprising 15 teeth irrigated with 5% NaOCl. This procedure was generated by a separate researcher (Y.A.T.) that was not involved in blinding and treatment procedures.

Blinding

In this study, it was not possible to mask the treating clinician due to the noticeable physical distinctions between various disinfecting irrigants, discernible by the dentist administering the treatment. Nevertheless, the patients and the assessors evaluating the reduction in bacterial count in the petri dishes were kept unaware of the specific irrigant employed.

Recruitment and eligibility criteria

Clinical examination to enroll eligible children was performed by an experienced principal investigator (A.M). Out of 49 patients who were assessed, 30 suitable participants were chosen for this study from a group of children aged between 3 and 5 years, encompassing both genders. They were referred for dental treatment to the Department of Pediatric Dentistry, Faculty of Dentistry, Damascus University. Inclusion Criteria included children who had not taken antibiotics in the past three months, with teeth showing no signs of internal root resorption, clinically necrotic teeth with pus secretion or fistula, and radiographic evidence of radiolucency around the root with absorption of one-third of the root or less. Children with systemic diseases that could affect their general health, symptomatic teeth, and teeth lacking sufficient bone support or with less than half of the root length remaining were excluded (Fig. 1).

Fig. 1.

Initial intra-oral image of one of the samples.

Preparing Pomegranate Peel Extract (PPE)

Ripe pomegranates were procured from the local market in Damascus City, Syria, in April 2023, with the plant’s identity verified by the Faculty of Agriculture at Damascus University.

The peels were manually separated from the seeds and sun-drieda for five days. Extraction and processing were conducted at the National Commission for Biotechnology, Damascus University, following the protocol outlined by Handa, Khanuja, Longo, and Rakesh²².

630 g of dried peel was ground using a mortar and pestle and subjected to extraction by soaking in 80% ethanol (supplied by the National Distillation Company) for 72 h. The process included daily filtration and solvent evaporation. The ethanol was subsequently removed under reduced pressure using a Delle Industrie rotary evaporator, yielding a dry extract with a yield percentage of 33.25%. The extract was air-dried and stored at − 20 °C for future use.

The study utilized a 5% w/v dosage (5 g of peel extract dissolved in 100 ml of distilled water), as described by Eltay et al.²³.

Cytotoxicity assessment of the obtained irrigants

Human Periodontal Ligament Fibroblasts for Cytotoxicity Testing of Irrigating Solutions.

Human periodontal ligament fibroblasts used in this study were obtained from the PDL of eight mandibular premolars extracted for orthodontic purposes. To minimize contamination, PDL tissue was carefully isolated from the middle third of the root, avoiding gingival tissue in the coronal portion. The cells were aseptically cultured in 75 cm² flasks containing 30 mL of Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with glutamine, 10% inactivated fetal bovine serum (FBS), 50 µg/mL streptomycin, and 3 µg/mL amphotericin.

The culture medium was refreshed every 2–3 days during the cell culture process, and the cells were passaged weekly. By the fourth passage, the cells had achieved sufficient confluence for cytotoxicity testing. They were subsequently plated in 60 mm culture dishes and maintained at 37 °C in a 5% CO₂ and 95% humidity environment until they reached confluence. At this stage, the culture medium was removed, and the cells were washed for five minutes using a solution containing FBS and ethylenediamine tetraacetic acid (EDTA). The cells were then trypsinized, with gentle agitation, to achieve separation.

Cytotoxicity of the irrigating solutions was evaluated 24 h post-incubation, using the Trypan blue assay to assess cell viability. Five replicates were prepared for each solution. Group A was treated with apple cider vinegar, while Group B received pomegranate peel extract. Trypan blue, a diazo dye, selectively stains dead cells blue. This staining occurs due to compromised cell membranes in dead cells, allowing the dye to penetrate, whereas live cells with intact membranes remain unstained. Consequently, dead cells appear blue under a microscope, and the method is often referred to as the dye exclusion technique.

After 24 h of incubation, the 10-well culture plates were removed from the incubator. Cells from the designated wells were collected using a multichannel micropipette, transferred to 1.5 mL Eppendorf tubes, and stained with 0.4% Trypan blue. The samples were gently mixed and left at room temperature for five minutes. A 10 µL aliquot of the stained cells was then placed on a Neubauer counting chamber, covered with a coverslip, and observed under a microscope. The numbers of viable (unstained) and dead (blue-stained) cells were counted and converted into percentages. The methodology in this pilot study was similar to that reported in previous studies^24,25.

The mean cell viability percentage was as follows: Apple Cider Vinegar (Group A): 79% and Pomegranate Peel Extract (Group B): 85%. This indicates that both irrigating solutions exhibit an acceptable level of cytotoxicity, according to the ISO 10993-5standard, which stipulates that an irrigant must achieve a cell viability level exceeding 70%²⁶.

Clinical intervention

A digital sensor (Vatech, Hwaseong, Korea) was used to capture periapical radiographs, providing detailed information on tooth anatomy, periapical lesion size, and canal curvature, aiding in the selection of appropriate teeth for the study. Enrolled patients were instructed to rinse with a 0.12% CHX (Aphamia, Hamah, Syria) before the procedure to reduce viable microbial content in dental aerosols.

The procedure was carried out under local anaesthesia using 2% lidocaine with 1:100,000 epinephrine (Novocol Pharma, Ontario, Canada) and rubber dam isolation (Sanctuary, Perak, Malaysia) (Fig. 2). The operative field and teeth were disinfected with 30% hydrogen peroxide followed by 10% iodine to minimize contamination during swab collection from the root canal. Decayed and soft tissues were removed with a low-speed handpiece equipped with a round carbide bur (RA#6, DENTSPLY Sirona, Charlotte, North Carolina, USA).

Fig. 2.

Shows isolation using a rubber dam.

Access cavities were prepared using a diamond fissure bur (Cylinder Flat End, FG, Coarse – 016, DENTSPLY Mailefer, Ballaigues, Switzerland) and a tungsten carbide bur (Endo-Z, DENTSPLY Mailefer, Ballaigues, Switzerland). The working length of each canal was determined using an apex locator (DENTSPLY Maillefer, Tulsa, Oklahoma, USA), and the Master Apical File (MAF), defined as the smallest file reaching the full working length, was identified. Subsequently, canals were filled with saline solution, then a sterile #15 hedströem file (Mani, Tochigi, Japan) in an in-and-out motion along the root canal dentine was done. Afterward, a sterile paper point (Gabadent, Guangdong, China) was inserted into the canal for 60 s to collect the first bacterial swab 1 (S1), for bacteriological analysis^1,27. The canals were then instrumented three sizes larger than the initial MAF (Fig. 3).

Fig. 3.

Shows the preparation of the canal walls after the initial swab is taken.

Irrigation process

Pomegranate Peel Extract (PPE) Group

Root canals were irrigated with 6 mL of 5% PPE between the three instrumentation files (IFs) 1 mm before the apical foramen using a 27-gauge irrigation needle (Henry Schein, Inc., New York, USA). Subsequently, root canals were irrigated with 4 mL of the 5% PPE after the last IF²⁴. Finally, 6 ml of distilled water was used as a final neutralizing irrigant in this group. The overall irrigation volume was 22 mL for each sample within 5 min.

Apple cider vinegar (ACV) Group

Root canals were irrigated with 6 mL of 5% ACV (Aldurra inc, Damascus, Syria) between the IFs 1 mm before the apical foramen using a 27-gauge irrigation needle (Henry Schein). Subsequently, root canals were irrigated with 4 ml of the 5% ACV after the last IF²⁴. Finally, 2 ml of 1% Sodium thiosulfate + 1% Tween 80 was used as a final neutralizing irrigant in this group²⁸. The overall irrigation volume was 22 mL for each sample within 5 min.

Sodium hypochlorite (NaOCl) group

Root canals were irrigated with 6 mL of 5% NaOCl (Canox, Damascus, Syria) between the IFs 1 mm before the apical foramen using a 27-gauge irrigation needle (Henry Schein). Subsequently, root canals were irrigated with 4 ml of the 5% NaOCl after the last IF²⁷. Finally, 2 ml of 1% Sodium thiosulfate was used as a final neutralizing irrigant in this group²⁸. (Fig. 4). The overall irrigation volume was 22 mL for each sample within 5 min.

Fig. 4.

Shows the irrigation of the canal.

Following the irrigation process in each sample, the canal was dried with paper points filled with saline solution, then a sterile #15 hedströem file in an in-and-out motion along the root canal dentine was done. Afterward, a sterile paper point (Gabadent, Guangdong, China) was inserted into the canal for 60 s to obtain the second bacterial swab (S2)²⁷. Subsequently, all canals were obturated using zinc-oxide eugenol cement and restored with glass ionomer cement.

Bacterial culturing

S1 and S2 glass test tubes of each sample were subjected to a vortex device (CSL-VORTEX, Thistle Scientific Ltd, Glasgow, United Kingdom) for 30 s to achieve a uniform bacterial suspension. Subsequently, 100 µl samples were extracted from the S1 and S2 bacterial suspension using a micropipette on a blood agar medium following a series of dilutions. The samples were then incubated in a bacteriological incubator (Bacteriological Incubator 6640-01-071–6596/National Appliance Heinicke Co. Tualatin, USA) under aerobic culture conditions at 37 °C for 48 h. Colonies Forming Units (CFU) were assessed in the samples, with each canal containing two petri dishes (one before irrigation (S1) and one after irrigation (S2)).

Afterward, the viable count of bacterial colony forming units (CFU) in 1 ml was determined using the following equation.

Number of bacteria/ml = number of CFU counted x Reciprocal of dilution factor x 10 (where the dilution factor adopted in counting dishes was 10⁻²).

The bacteria units’ number was converted into logarithmic numbers to facilitate bacterial colonies counting and statistical analysis.

Statistical analysis

One-Way ANOVA and Chi-square tests were used to assess the distribution of children in the sample regarding age and gender into the groups respectively. The normal distribution of quantitative data was assessed using the Shapiro-Wilk test. The results showed that decimal logarithm of the bacterial colony values in NaOCl group after irrigation had an abnormal distribution, while PPE and ACV groups displayed a normal distribution before and after irrigation. Intra-group comparisons were made using Paired sample t-test for PPE and ACV groups, and Wilcoxon signed ranks test for NaOCl group. Between-groups comparisons were made using One-Way ANOVA, Kruskal-Wallis, Mann-Whitney U, and Independent Samples tests. Statistics were calculated using Statistical Package for Social Sciences (SPSS) version 25.0 (IBM, Chicago, IL, USA). The significace level was set at α = 0.05

Results

A total of 45 necrotic incisors in 30 patients (16 males and 14 females) aged between 3 and 5 years ( = 4.5) were included in the study. No significant differences were reported between the groups regarding the age (p = 0.707) and gender (p = 0.715) of the treated patient, indicating that the allocation of patients into study groups was randomized. Age and gender distributions across groups and statistical tests’ results for their comparison among groups are presented in Table 1 and ​ 2 respectively.

Table 1.

Descriptive and analytic statistics of age distribution across groups.

Group	Mean	standard deviation	Minimum	maximum	p-valuea
Pomegranate Peel Extract	5.72	0.51	5.00	6.5	0.707a
Apple Cider Vinegar	5.56	0.44	5.00	6.3
Sodium Hypochlorite	5.75	0.50	5.00	6.5

a One-Way ANOVA Test.

It is worth noting that no samples were excluded during laboratory procedures and bacterial culturing, and no cases of post-treatment flair-up or discomfort were recorded.

Table 2.

Descriptive and analytic statistics of gender distribution across groups.

Group	Gender		Total	p-valuea
	Boys	Girls
Pomegranate Peel Extract	5	6	11	0.715a
Apple Cider Vinegar	7	4	11
Sodium Hypochlorite	4	4	8
Total	15	15	30

a Chi-square’s Test.

Intra-group results of aerobic bacterial culture for study samples

Figures 5 and 6, and 7 presents the bacterial culture procedures before and after irrigation procedures among the groups in aerobic conditions.

Fig. 5.

Bacterial culture procedures in aerobic conditions in a sample of PPE group: (A) before irrigation, and (B) after irrigation.

Fig. 6.

Bacterial culture procedures in aerobic conditions in a sample of ACV group: (A) before irrigation, and (B) after irrigation.

Fig. 7.

Bacterial culture procedures in aerobic conditions in a sample of NaOCl group: (A) before irrigation, and (B) after irrigation.

Table 3 summarizes the evaluation of decimal logarithm of the bacterial colony counts among the groups.

Table 3.

Descriptive statistics of CFU count, reduction percentage, and mean decrease amount in CFU count before and after irrigation among the groups.

Irrigant	Studied Stage	Mean ± Standard deviation	Range	Reduction percentage	Mean of decrease amount in CFU count	T-value	*p-value
Pomegranate Peel Extract	Before irrigation	5.59 ± 0.269	5.00–5.90	60.43%	3.38 log10 CFU/mL	29.729	< 0.001
	After irrigation	2.21 ± 0.37	1.40–3.00
Apple Cider Vinegar	Before irrigation	4.36 ± 0.431	3.54–4.97	51.38%	2.15 log10 CFU/mL	11.656	< 0.001
	After irrigation	2.20 ± 0.58	2.89–3.05
Sodium Hypochlorite	Before irrigation	4.85 ± 0.257	4.36–5.19	87.49%	4.23 log10 CFU/mL	− 3.408	< 0.001
	After irrigation	0.61 ± 0.15	0.00–2.20

*Paired sample t-test for PPE and ACV groups, and Wilcoxon signed ranks test for NaOCl group.

From the previous table, it’s observed that PPE contributed to a reduction in the count of bacteria by an average reduction of 3.38 log¹⁰ CFU/mL with a statistically significant difference (p < 0.001). Moreover, ACV contributed to a reduction in the count of bacteria by an average reduction of 2.15 log¹⁰ CFU/mL with a statistically significant difference (p < 0.001). Finally, NaOCl contributed to a reduction in the count of bacteria by an average reduction of 4.23 log¹⁰ CFU/mL with a statistically significant difference (p < 0.001).

Between-groups results of aerobic bacterial culture for study samples

One-Way ANOVA revealed that the three groups showed no statistically significant differences in decimal logarithm of the bacterial colony values before irrigation (S1) step (p = 0.846), but Kruskal-Wallis test exhibited statistically significant differences in after irrigation (S2) step (p < 0.001). Tables 4 and 5 present the pairwise comparisons.

Table 4.

The results of the pairwise comparisons between NaOCl, PPE, and ACV groups after irrigation.

irrigation solution A	irrigation solution B	Mann-Whitney U Value	Z-value	*p-value
Sodium hypochlorite	Apple cider vinegar	22.500	−3.804	< 0.001
	Pomegranate peel extract	14.500	−4.143	< 0.001

* Mann-Whitney U test.

Table 5.

The results of the pairwise comparisons between ACV and PPE after irrigation.

irrigation solution A	irrigation solution B	T-value	*p-value
Apple cider vinegar	Pomegranate peel extract	−0.15	0.988

* Independent Samples test.

Mann-Whitney U test revealed that there is significant difference between the groups after the irrigation process. NaOCl has higher decimal logarithm of the bacterial colony values than PPE and ACV groups.

Independent Samples Test revealed that there is no significant difference between the two groups after the irrigation process.

Discussion

The increasing prevalence of oral diseases globally, particularly in developing nations, along with the growing resistance of pathogenic bacteria to existing antibiotics and chemotherapies, as well as opportunistic infections in immunocompromised individuals, underscores the demand for safe, efficient, and cost-effective alternative preventive and therapeutic solutions. This need is further compounded by financial constraints in developing countries^29,30.

Punica granatum has antiviral, bactericidal, and antifungal properties due to the immune modulation and anthelminthic actions of pomegranate³¹. The polyphenols and tannin extracts found in Punica granatum are effective in preventing biofilm-related oral diseases like dental caries, gingivitis, and periodontitis. Therefore, it is likely that Punica granatum can also target endodontic biofilms^23,31–33. The study of Gallas et al.³⁴highlighted that pomegranate peel extract exhibited antimicrobial and antioxidant qualities against resilient root canal pathogens. These findings suggest that it could serve as a promising adjunct treatment in root canal therapy. Moreover, a study has suggested that Punica Granatum is a suitable transport medium for avulsed teeth. The findings revealed that 1% Punica Granatum juice was as effective as HBSS in preserving PDL cell viability, indicating that Punica Granatum positively affects fibroblast cells³⁵. The aforementioned properties beside the cytotoxicity pilot study results encouraged the use of this irrigant in the present study to assess its clinical effectiveness.

Apple cider vinegar was presented as an antibacterial agent in root canals, in addition to its ability to remove the smear layer. Therefore, it was suggested to assess its use as an alternative irrigation fluid in endodontic treatment^36,37. A study comparing the cytotoxicity of ACV and NaOCl revealed that NaOCl was more cytotoxic than ACV. The findings indicate that while ACV demonstrates promise as an alternative irrigation solution due to its antibacterial properties, its cytotoxicity at higher concentrations raises concerns regarding its safety in root canal treatments. However, the current cytotoxicity pilot study showed acceptable cell viability cells. Accordingly, further research is required to comprehensively evaluate its effects and develop guidelines for its safe and effective use in dental practice³⁸. This suggests that ACV may be a potential candidate for testing in irrigation procedures.

Hence, the effectiveness of 5% Pomegranate peel extract and 5% apple cider vinegar as irrigants was investigated.

A search in the literature showed that this clinical study is the first one to evaluate the efficacy of irrigation with Pomegranate peel extract and apple cider vinegar in disinfecting the necrotic root canals.

It was compared with a 5% sodium hypochlorite solution in this research as the control group, given its status as the gold standard for irrigation in endodontic treatment¹.

To ensure standardization of specimens and to control for anatomical differences, anterior teeth were chosen for the study. Their relatively straight root canal anatomy reduces variations in canal morphology, which may differ among the canals of multirooted teeth³⁹.

The study employed manual preparation with hand files, ensuring that apical preparation extended three measurements beyond the main file in all samples to minimize bacterial presence within the canal^1,40.

A 2% solution of sodium thiosulfate was employed post-sodium hypochlorite irrigation as a neutralizing agent, and 1% Sodium thiosulfate + 1% Tween 80 was used as a final neutralizing irrigation solution in ACV Group. This was done to eliminate any residual ion traces from the solution under study, thereby preventing any interference with the outcome of the bacterial culture and avoiding false negative results^28,41.

Nonetheless, extensive irrigation with distilled water was implemented to eliminate any remnants of Pomegranate peel extract. This was necessary as the neutralizing agent for Pomegranate peel extract had not been previously utilized in clinical studies involving endodontic treatment in patients.

Bacterial samples were obtained using sterile paper points and a sterile Hedströem file, which were manoeuvred in an in-and-out motion along the root canal. This method was employed to procure a more accurate bacterial sample, as relying on paper points for sampling has constraints. Specifically, sampling with paper points alone only captures microorganisms within the root canal space, overlooking those residing inside the dentine tubules⁴².

Based on the findings of the study, the tested null hypothesis was rejected. The study’s findings indicated that initial bacterial counts were consistent across experimental groups before irrigation, and post-irrigation bacterial counts showed a decrease in CFU count compared to pre-irrigation counts, irrespective of the irrigation solution used.

In our Study, the average percentage of CFU count reduction in the bacterial population in the PPE Group reached 60.43% and 51.56% in the ACV group, showing statistically significant variances between the study’s phases.

The findings of this study were in accordance with the results of Mallwika et al., which evaluated the antimicrobial efficacy of pomegranate extract, sodium hypochlorite, chlorhexidine, and other herbal groups against Enterococcus faecalis, Fusobacterium nucleatum, and Staphylococcus epidermidis. The findings showed that pomegranate extract exhibited a notable antibacterial effect against E. faecalis⁴³.

These results are also consistent with a study conducted by Mallya et al. in 2019, which demonstrated that the combination of Punica granatum with sodium hypochlorite and CHX yielded superior outcomes compared to when these irrigants were used individually or when hypochlorite and CHX were combined. This points to the notable antimicrobial activity of Punica granatum¹⁸.

These findings could be attributed to the phenolic compounds in PPE, which create difficulties for microorganisms to survive by disrupting bacterial cell walls. Additionally, the antioxidant properties of anthocyanins in pomegranate are highly bioactive. Combining PPE with hypochlorite and CHX may enhance the antibacterial properties, as suggested by studies such as those by Reddy et al.¹⁷ and Vasconcelos et al.⁴⁴.

Our results are consistent with a study revealed that PEE was effective against S. mutans which constitute about 16% of the microorganisms in the root canals^1,36.

This may be attributed to the disruption of polysaccharide synthesis caused by the presence of ellagitannin and punicalagin, which hinder the adherence mechanism of these organisms to the dental surface. Furthermore, the tannin in PPE can penetrate the microorganisms’ cell wall, binding to its surface and causing protein precipitation. This action suppresses enzymes like glycosyl transferase, which is crucial for carbohydrate breakdown and provides nutrition to the organisms^18,45.

These findings also align with a study by Mohanty et al.²¹, which compared the antibacterial properties of ACV against Enterococcus faecalis. The study concluded that the antibacterial efficacy of apple cider vinegar was comparable to that of 5% NaOCl against Enterococcus faecalis, a highly resistant bacterium known for its role in endodontic treatment failure^46,47.

Moreover, these results are consistent with an in-vitro study that assessed the antibacterial efficacy of ACV as a root canal irrigant using the Endovac irrigation system. The Endovac system effectively eliminated E. faecalis from root canals when using either NaOCl or ACV⁴⁸.

Our findings align with a study by Alyamany et al.⁴⁹, which investigated the antibacterial effectiveness of various irrigants, including ACV and NaOCl on primary teeth. ACV demonstrated acceptable antibacterial activity against faecal intestinal bacteria in line with our study’s results.

While our study partially concurs with an in-vitro investigation comparing the irrigation efficacy of 5% ACV with a 3% NaOCl solution, it differed in terms of statistical significance when compared to NaOCl. This disparity may be attributed to the lower concentration of the NaOCl (3%) used in the study and the controlled laboratory setting, where the virulence of microbes and biofilms may differ from clinical conditions⁵⁰.

Our findings in the NaOCl group are consistent with the research conducted by Alkhourbotly et al.⁵¹, which compared the antibacterial efficacy of QMix and AgNP with NaOCl in primary molars root canals. Their study demonstrated an 89% reduction in the NaOCl group.

PPE and ACV demonstrated potent antibacterial properties. However, further clinical studies with extended follow-up periods are required to assess and compare radiological and clinical treatment outcomes.

A notable limitation of this study is the absence of a follow-up period to evaluate these outcomes comprehensively. Future research should incorporate follow-up evaluations to provide a more thorough comparison of clinical and radiographic treatment effectiveness.

Conclusion

Although sodium hypochlorite has shown effectiveness in reducing bacterial counts, Pomegranate peel extract and Apple cider vinegar have also displayed some antibacterial activity. However, their overall efficacy is less established, which raises questions about their viability as a natural alternative to a 5% sodium hypochlorite solution.

Author contributions

A.M conceptualized the idea, provided the clinical and in vitro work, and contributed to the writing and documenting; M.L and H.A conceptualized the idea and supervised the research. S.A.A. contributed in bacterial culture. Y.A.T interpretation of data and the revision, formatting, reediting of the manuscript and conducted the statistical study. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Damascus University (funder No. 501100020595).

Data availability

De-identified data are available upon reasonable request to the corresponding author.

Declarations

Consent for publication

Informed consent was obtained from all children’s parents for the data publication of as part of this study.

Competing interests

The authors declare no competing interests.

Human Ethics and Consent to Participate declarations

The Research Ethics Committee of the Faculty of Dentistry, Damascus University ethically approved this human-research project (Approval number: (UDDS-2653-14022023/SRC-365)).

Informed consent

was obtained from all children’s parents to participate in this trial.