This was an in-vitro study conducted at the Department of Conservative Dentistry and Department of Microbiology, College of Dentistry/University of Duhok, Iraq, from January 2020 till June 2020.

The method of herbal extraction was as follows: 200 gm of Miswak (S.persica) was cut into irregular pieces and dried in an oven at a temperature of 45°C±5°C for a period of 24 hours until it became completely moisture free and then was grounded by an electric miller (high speed multi-functional crusher) to form a fine powder [12]. A 200 gm of green tea leaves and German Chamomile were also separately grounded into powder form. Then, 150 gm of each herbal powder was soaked in 300 mL of 96% ethanol inside a flask and kept at room temperature [13,14]. After 72 hours, the solutions were filtered using a filter paper. The ethanol was evaporated and the extracts were concentrated using a rotary flask evaporator. These extracts were stored at 4°C in refrigerator until need [14].

Exclusion and Inclusion criteria: A total of 75 extracted human mandibular of first and second primary molars were collected from the polyclinic dental center and a private clinic and were used for this study. For the purpose of this study, primary molars, with atleast 2/3^rd remaining root, which were extracted due to the presence of chronic abscess and/or sinus tract were chosen. Any tooth with root caries, more than two-thirds loss of root structure or a patient with antibiotic use for systemic diseases in the past 4 weeks were excluded from the study.

Herbal extraction: Teeth debris and soft tissue remnants was cleansed using ultrasonic scaling device (ACTEON, Newtron, France). The anatomical crown of all the teeth were amputated at CEJ perpendicular to the long axis of the teeth by a water cooled diamond disc bur (Tompet, Norderstedt, Germany) and the roots were separated. Only the distal roots were used in the study. The exploration of the radicular canal was accomplished with size 15-K file (NIC, England, United Kingdom). The working length of roots measured (10±1) mm. All teeth were immersed in normal saline until required [15].

Protaper system rotary files were used to prepare the root canal biomechanically to size F3 (30/9%) (Dentsply-Maillefer, Ballaigues, Switzerland). The canals were irrigated with 0.5% NaOCl (CERKAMED, Poland) for disinfection of the canals and final rinse with normal saline was done [14].

The apical foramen of each root sample was sealed by applying flowable composite (Harvard, GmbH, Germany) on the root apex to prevent fluid leakage during microbiological study. Each root was embedded in silicon impression material block-up and 2 mm of cervical margin was exposed to facilitate the grasping and holding of root samples [Table/Fig-1]. The specimens were wrapped with aluminum foil and then autoclaved (mocom, Italy) at 121°C for 30 minutes [16].

[Table/Fig-1]:

Samples embedded in silicone impression material.

The samples were divided randomly into five groups of 15 teeth for each. In Group 1, 0.5% NaOCl was used as a positive control root canal irrigant. While in Group 2, 0.9% normal saline was used as negative control. Group 3, 4 and 5 were irrigated with miswak extract, green tea extract and chamomile, respectively.

The standard strain of E.faecalis (ATCC^®29212^™), obtained from Media Laboratory Private (Hawler/Iraq), was prepared by harvesting (4-5) colonies with a previously sterilized circular loop and dissolved into a glass container containing 3 mL Brain-Heart Infusion (BHI) broth. The cultures were grown overnight at 37°C in the BHI broth and the growth was determined by change in turbidity after 24 hours [17]. A volume of 10 μL of suspension was inoculated into each tooth and incubated for 72 hours at 37°C to allow growth of E.faecalis inside the root canals. The specimens were handled using flamed tweezers to prevent contamination [18].

Canals were filled with 2 mL of the corresponding irrigant in each group and were permitted to stay for 10 minutes. The root canals were then rinsed with 2 mL of saline for 1 minute. Sterile paper point (size F3) (DIADDENT, Korea) was left in the wet canal for 1 minute and was transferred into a test tube containing 1 mL sterile normal saline for the first tube and 0.8 mL normal saline for the other 5 tubes to form (1-6) tubes for the serial dilution. After mixing well 0.2 mL of tube number 1 had been transferred to tube number 2 and the same procedure was done for the another tubes. From this 1 mL of each dilution 100 μL was pipetted onto a sterile Mueller-Hinton agar plate. These plates were incubated for 24 hours at 37°C in an incubator. After incubation, the colonies were counted under good illumination by using camera zoom (mobile phone telescope, 8X, China) [Table/Fig-2]. Then the number of the colonies was multiplied by the dilution factor to get Colony Forming Unit (CFU) [19].

[Table/Fig-2]:

Culture plates show the colonies of bacteria on a Mueller-Hinton agar.

Sensitivity test: A 0.1 mL of BHI broth of E.faecalis was cultured on Mueller-Hinton agar plates. Wells of 5 mm diameter were made in the agar surface. Then tested materials were added to these wells. After 24 hours of incubation at 37°C, the diameter of inhibition zones were measured [20].

The comparison of colony counts among study groups was examined by using Kruskal-Wallis one-way Analysis. Statistical Package for the Social Science (SPSS) version 26.0 was used. Student-Newman-Keuls Method used for the comparison of colony counts between positive and negative controls and other study groups. A p-value less than 0.05 were considered significant.

The total number of CFU/mL obtained from the experimental groups after 72 hours of incubation is presented in [Table/Fig-3] and comparison between study groups is presented in [Table/Fig-4]. Antibacterial activity of green tea extract was most effective against E.faecalis among the herbal experimental groups followed by miswak and chamomile respectively. No statistically significant difference (p>0.272) was found between the green tea extracts and between NaOCl. NaOCl showed maximum antibacterial efficacy against E.faecalis, while the antibacterial property of 0.9% normal saline had the least effect against E.faecalis. The results of inhibition zone of sensitivity test are presented in [Table/Fig-5].

Primary teeth are essential in the life of the child as they help chew, speak, contribute to esthetics, maintain the integrity of the dental arches and finally lead the teeth to their correct position [5]. In early childhood, carious teeth are not only predictive of potential dental problems, but can adversely affect development and cognitive growth by affecting nutrition [21].

Effective endodontic treatment of primary teeth relies on the eradication (if present) of microbes from the root canal region and the prevention of re-infection [5]. The root canal treatment is done under continuous irrigation with hand or rotary instrumentation to extract the inflamed and necrotic tissues, microbes/biofilms and other root-canal space debris [22]. Due to its high prevalence in secondary endodontic infections, E.faecalis was selected for this study [23].

Optimal irrigation is based on the usage of a mixture of two or more irrigants in a particular series to meet the aim of safe and efficient irrigation [22]. Despite its unpleasant odour and taste for the child and the possible risks of swallowing and poisoning during the procedure, the most commonly used irrigant solution is NaOCl, which also has burning properties and lacks differentiation between necrotic and vital tissues when in contact with apical and periapical tissues [24]. It also has adverse effects on dentin, such as lowering its flexural strength and elastic modulus. NaOCl toxicity to vital periodontal tissues, risk of emphysema when overfilling, allergic potential had been demonstrated [14]. Risk of damage to permanent tooth follicles, peripheral tissue and oral mucosa, through inappropriate use of NaOCl has been noted in paediatric endodontic patients [24].

Common used concentration of NaOCl range from 0.5-5.25%, so to reduce the cytotoxicity of NaOCl especially in paediatric dentistry, 0.5% NaOCl was used as positive control in this study. Herbal extracts were used to disinfect the root canal in primary teeth to overcome the undesired effects of NaOCl. Herbal medicinal products gain importance due to their therapeutic properties and biocompatibility [9]. The present study evaluates antibacterial efficacy of herbal irrigants in comparison with 0.5% NaOCl which is commonly used in concentrations ranging from 0.5% to 5.25%. Hypochlorite is reported to destroy the target microorganisms within seconds even at low concentrations [25].

Studies have compared the biological effects of mild and strong NaOCl solutions and demonstrated greater cytotoxicity and caustic effects on healthy tissue with 5.25% NaOCl than with 0.5% and 1% solutions [2]. The relationship between the concentration and cytotoxicity of NaOCl had been reported by Chang YC et al., 2001. Therefore, it is recommended to replace 5.25% of NaOCl by 0.5-1% NaOCl for canal irrigation [26]. For this study, 0.5% NaOCl tested as positive control. NaOCl showed highest antibacterial effect (79×10² CFU) [Table/Fig-3] against E. faecalis compared to herbal extracts, while normal saline had the least (1974×10² CFU) [Table/Fig-3].

In the present study, authors found that green tea showed a good antibacterial activity against E. faecalis compared to the control group with (141×10² CFU) [Table/Fig-3]. Green tea has active physiological materials that have great curing, antioxidant action, anti-inflammatory, important scavenging properties that are all helpful to make it ideal for intracanal irrigation. Other good features of green tea, as a cleaning agent, include accessibility, affordability, low toxicity and long shelf life [27].

The current study agrees with Ramezanali F et al., who demonstrated that green tea showed acceptable antibacterial effect on the biofilm of E. faecalis [14]. Nonsignificant results were obtained when CFU in green tea and NaOCl group were compared (p>0.0001). Similarly, the results of this study are compatible with the American Medical Association which showed that green tea has excellent medicinal values. It is also observed that green tea has antibacterial effect against E.faecalis [28]. Also, Jerin J et al., showed that 2.5% NaOCl had the best antimicrobial property against E. faecalis, followed in descending order by green tea extract, garlic extract and neem leaf extract [29].

Miswak (S. persica) showed antibacterial effect against E.faecalis with colony count (932×10²CFU) [Table/Fig-3]. The antimicrobial and cleaning effects of Miswak are attributed to different chemicals that can be detected in its extracts. Other effects are thought to be attributable to the presence ofcyanogenic glycoside and benzyl-isothiocyanate and to its high content of NaCl and KCl, as well as Salvadourea and Salvadorine, saponins, tannins, vitamin C, silica, certain naturally occurred anionic components and resin [30].

But results of this study disagreed with Al-Azzawi A, that used agar diffusion methods to compare between antibacterial effect of S.persica and green tea against E.faecalis. The study showed S.persica more effective against E.faecalis than green tea [20]. German Chamomile (Marticariarecutitia L.) extract also showed antibacterial effect against E.faecalis with number of colony (1074×10² CFU) [Table/Fig-3] as a root canal irrigation in primary teeth. A few studies discussed the antibacterial effect of chamomile as intracanal irrigation.

Vakil N et al., observed that chamomile oil exhibited antibacterial activity against gram-positive bacteria such as Bacillus subtilis, Staphylococcus aureus, Streptococcus mutans, and Streptococcus salivarius at a concentration of 25 mg/mL, but at 150 mg/mL concentration, chamomile exhibited no microbial effect against E. faecalis and C. albicans. However, chamomile efficacy is not more than 2% Chlorhexidine, antimicrobial effect against both microorganism had been reported at higher concentration 250 mg/mL [31].

One particular component of the irrigant solution could be attributed to the anti-inflammatory effect. In the German chamomile irrigant solution salicylic acid in the form of a methyl ester provides an anti-inflammatory effect [32]. Flavonoids, including apigenine, chamazulene and α-bisabolol are the other constituents found in whole plant chamomile extract. The flavone act as anti-inflammatory agent which can significantly affect bacteria [33].

In the present study, there was no significant difference statistically between NaOCl and green tea. NaOCl shows the highest antibacterial effect against E.faecalis followed by green tea, miswak and chamomile respectively. Some differences between the results of other studies and our results might be due to the differences in the method of extract preparation, the quality of (green tea, Miswak and Chamomile) and culture time.

Ample options to choose specific types of herbs were not available. Only herbs of local origin were obtainable. The applied methods of preservation of herbal materials before being used in this study could be questionable. For sensitivity test, both disc and wells should have been used.

Herbal irrigant solutions showed acceptable antibacterial effect against E. faecalis in primary teeth. Green tea specifically showed no significant difference with NaOCl, whereas significant differences were observed with miswak and chamomile. To avoid undesirable properties of NaOCl, the use of herbal substitutes as intracanal irrigant solutions, particularly in paediatric dentistry, should be considered.