Antibacterial Effect of Matricaria chamomilla L. Extract Against Enterococcus faecalis

Introduction

Root canal debridement is important for successful endodontic treatment.¹ Root canal irrigants should have the following attributes: low toxicity; antimicrobial effect; non-caustic to periodontal tissues; non-allergenic; and the ability to dissolve tissue and root canal debris, inactivate endotoxins, lubricate the canal, and remove the smear layer.²

Sodium hypochlorite, a root canal irrigant that is widely used in endodontic therapy, has antibacterial qualities and assists in mechanical root canal debridement and pulp dissolution.³ In combination with ethylenediaminetetraacetic acid (EDTA) or citric acid, sodium hypochlorite can remove the smear layer; however, it also has toxic effects, allergic potential, and a bad taste.^4,5

Chlorhexidine gluconate can also be used as an endodontic disinfectant, because it can penetrate the bacterial wall and attack the cell cytoplasm. It has an antimicrobial effect, does not irritate the periapical tissue, and does not have a bad taste, but it does not disintegrate pulp tissue.⁶

Despite the use of various instruments and irrigators during endodontic treatment, complete debridement is not possible due to the anatomical complexity of the root canal system.⁷

Research has proven that Enterococcus faecalis can be isolated in 24–77% of endodontically treated canals,⁸ because of its resistance to intracanal drugs and its ability to form biofilm, invade dentinal canals, and survive in the absence of nutrients for long durations.^9,10

The antibacterial effect of root canal disinfectants may be reduced in vivo by the presence of organic compounds (eg, albumin) in the inflammatory exudate and hydroxyapatite (the main inorganic component of dentin).¹¹

The problems of drug-resistant microorganisms and drug side effects have encouraged investigators to find alternative solutions that increase the antimicrobial effect with no toxicity.

Nowadays herbal products are gaining popularity in dental and medical practice.

Matricaria chamomilla is a well-known medicinal plant from the Asteraceae family. The phytochemical composition of M. chamomilla essential oil and extracts has been identified as containing more than 120 constituents.¹² M. chamomilla has been shown to have strong antibacterial potential against Gram-positive and Gram-negative bacteria.¹³ The chamomile plant is known to have antibacterial, anti-inflammatory, antiviral, and antioxidant effects, due to the presence of α-bisabolol, luteolin, quercetin, and apigenin. The clinical efficacy of chamomile has also been reported in selectively removing the root canal smear layer.¹⁴

Therefore, the aim of this in vitro study was to evaluate the M. chamomilla ethyl acetate extracts as a potential antibacterial agent in the inhibition of E. faecalis in comparison with sodium hypochlorite and chlorhexidine (CHX).

Materials and Methods

This paper has been approved by the Ethics Commission of Medical Faculty, University of Prishtina “Hasan Prishtina” (Nr. 2417). For this study, we used 100 extracted human single-rooted teeth in order to collect dentine powder. Dentine powder was used to assess inhibitory effect in medications. Teeth were extracted for periodontal and orthodontic reasons. Patients have given written consent that their teeth after extraction can be used for research purposes in the future. This consent has been approved by the same Ethics Commission. Before the experiment, the teeth were stored in 1% sodium hypochlorite solution and then rinsed and autoclaved at 121℃ for 15 min. The crowns of the teeth were cut at the enamel–cementum junction with a diamond saw (Smart Cut 4002, UKAM, Valencia, CA, USA). The root canal was accessed and the pulp tissue was removed. We then used the root canal instruments Endostar E5 (Poldent Co., Warsaw, Poland) to enlarge the root canal and to collect dentin powder. The collected dentin powder was suspended in distilled water at a concentration of 28 mg per 50-μL aliquot.

Materials used in this study were as follows: 2% sodium hypochlorite (ChloraXD, Cerkamed, StatowaWola, Poland), 2% CHX (Gluco-Chex, Cerkamed), ethyl acetate (EthOAc; Sigma Aldrich, Switzerland), and dimethyl formamide (DMF; Sigma Aldrich). Dimethyl formamide was used as extract solvent with no inhibition effect.

The M. chamomilla plants were collected in a Kosovo field in 2018. The plant was preserved in the herbarium of the Biology Department of University of Prishtina (no. 324/2018). The aerial part of the plant was air-dried and ground to a powder with a blender. The dried powder was vacuum-packed and stored at –20℃ until use. Dried M. chamomilla powder (10 g) was extracted twice with 100 mL of 80% ethyl acetate (EthOAc) with a Soxhlet apparatus at boiling point, and the extract was concentrated with a rotary evaporator at 40℃ (Figure 1). This extract A4(5) was stored in a deep freeze until the time of the experiment. For the antibacterial activity assays, the extracts were dissolved in N,N-dimethylformamide (DMF) at a concentration of 100 mg/mL and stored at 4℃ as a stock solution. Enterococcus faecalis (ATCC 29212 Thermo Fisher Scientific) was used to evaluate the antibacterial potency of the plant extract, the irrigants, a mixture of the plant extract with the endodontic irrigants, and all of the above mentioned materials with dentin powder. The antibacterial activity of the Soxhlet plant extracts and the endodontic irrigants was analyzed using a disk diffusion assay. A suspension of E. faecalis was cultured for 48 h in 1 mL of sterile brain heart infusion (BHI, Liofilchem, Roseto degli Abruzzi, Italy) at 37℃, then adjusted to a turbidity of 0.5 on the McFarland scale (1.5 × 108 cells/mL). This culture was used throughout the experiments. Mueller–Hinton agar (10 mL) was poured into Petri dishes according to manufacturer instructions, which were then inoculated with strains of bacteria by the addition of 0.1 mL of cell culture medium. Sterile filter paper disks impregnated with the endodontic irrigants, plant extracts, and the combination of endodontic irrigants and plant extracts, and all of the above mixed with dentin powder (10 mg/mL), were placed on top of Mueller–Hilton agar plates. The plates were incubated at 5℃ for 2 h to permit diffusion of the plant extract, and then incubated at 35℃ for 24 h. Disks loaded with the extract solvent (EthOAc) were used as a control group. Vernier calipers were used to record the inhibition zones.

Figure 1 Plant extracts-extracted with a Soxhlet apparatus.

To evaluate the antibacterial effect of the medicaments on dentin powder, 50 μL aliquots of the dentin powder suspended in water were mixed and incubated with 50 μL of the medicaments in sealed test tubes at 37℃ for 1 h or 24 h, before the addition of bacteria. Control groups consisted of 50 μL of sterile water instead of dentin powder. The total volume of the test and control mixtures was 150 μL. All mixtures were incubated at 37℃. The dentin powder/endodontic irrigant/bacteria suspension was mixed with a sterile pipette twice (before the 1-h sample) or three times (before the 24-h sample) during the incubation time. Samples for bacterial culturing (10 μL per sample) were taken from the experimental and control suspensions at 5 min, 1 h, and 24 h after the addition of E. faecalis. The Petri dishes with agar were observed after 5 min, 60 min, and 24 h (Figure 2). The results were recorded based on the standard disk method according to the US Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antibiotic Sensitivity Testing (EUCAST).

Figure 2 Inhibition zone (mm) of used agents.

The recorded data were analyzed with SPSS Statistics 22.0. One-way ANOVA was used to test for normal distribution of the tested groups, and the Kruskal–Wallis test was used for non-normal group distribution. A level of P<0.05 was considered to be statistically significant.

Results

The growth of E. faecalis colonies was recorded at 5 min, 60 min and 24 h after the addition of an ethyl acetate extract of M. chamomilla - A4(5) to all test samples with endodontic irrigants without dentin powder, and to all samples of the same mixture with dentin powder. At 5 min after the combination of A4(5) with sodium hypochlorite without dentin powder there was no inhibition zone. When the extract was used alone, the inhibition zone was 9.7 mm, and in combination with CHX it was 30.3 mm. When the M. chamomilla ethyl acetate-extract was used alone, the inhibition zone was 31.7 mm. There was a significant difference at 5 min after the culture (E. faecalis) was inoculated in the samples without dentin powder according to the tested irrigants (P=0.0015) (Table 1).

Table 1 Effect of EthOAc Extract of Matricaria chamomilla with and without Irrigant in Enterococcus faecalis Growth without Dentine Powder

At 5 min after culture inoculation, the samples with dentin powder exhibited no inhibition zone in the A4(5)/sodium hypochlorite group. However, when the plant extract was used alone, the inhibition zone measured 7.3 mm, and after combination with CHX it measured 25.3 mm. When sodium hypochlorite was used alone, the inhibition zone was 26.0 mm. Thus, there was a significant difference after the addition of dentin powder in all groups, depending on the medicament used (P=0.0015) (Table 2).

Table 2 Effect of EthOAc Extract of Matricaria chamomilla with and without Irrigant in Enterococcus faecalis Growth with Dentine Powder

At 60 min after culture inoculation, the samples without dentin powder exhibited no inhibition zone in the extract/sodium hypochlorite group. However, when the extract was used alone, the inhibition zone was 9.3 mm and in combination with CHX, it was 25.7 mm. Additionally, when sodium hypochlorite was used alone, the inhibition zone was 34.7 mm. There was a significant difference at 60 min after inoculation of the culture (E. faecalis) in samples without dentin powder according to the tested irrigants (P=0.0015) (Table 1).

At 60 min after culture inoculation, the samples with dentin powder exhibited no inhibition zone in the extract/sodium hypochlorite group. When the extract was used alone, the inhibition zone was 7.7 mm, after combination with CHX it was 21.0 mm, and when sodium hypochlorite was used alone it was 18.0 mm. Therefore, there were significant differences after the addition of dentin powder after 60 min in all groups, depending on the medicament used (P=0.0015) (Table 2).

At 24 h after culture inoculation, the samples without dentin powder exhibited no inhibition zone in the extract/sodium hypochlorite group. However, when the extract was used alone, the inhibition zone was 9.7 mm, and in combination with CHX, it was 25.3 mm. Additionally, when sodium hypochlorite was used alone, the inhibition zone was 38.3 mm. There was a significant difference at 24 h after culture inoculation (E. faecalis) in samples without dentin powder according to the tested irrigants (P=0.0015) (Table 1).

At 24 h after culture inoculation, the samples with dentin powder exhibited no inhibition zone in the extract/sodium hypochlorite group. When the extract was used alone, the inhibition zone was 7.7 mm, after combination with CHX it was 21.7 mm, and when sodium hypochlorite was used alone it was 14.3 mm. There was a significant difference after the addition of dentin powder after 24 h in all groups depending on the medicament used (P=0.0015) (Table 2).

One-way Anova was used to analyse intergroup comparison. There was no statistical difference between group treated with EthOAc extract of Matricaria Chamomilla and group treated after mixing EthOAc extract with dentine powder (F=17.682, p=0.014).

But, there was statistical difference between the group treated with EthOAc/CHX and the group treated with EthOAc/CHX after mixing with dentine powder (F=82.14, p<0.001).

Discussion

E. faecalis has several virulence factors that influence its survival in the hostile root canal environment: secretory factors, adhesins, surface structures such as capsular polysaccharides, and antibiotic resistance.¹⁵ As an endopathogenic microorganism, E. faecalis has the capacity to penetrate into the dentin tubules and to adhere to the surface of the dentin.¹⁶ For these reasons, this microorganism was selected for testing in our research.

The disk diffusion method for evaluating the antimicrobial activity of the experimental materials was selected because it is a quick and simple procedure that allows for direct contact of the material with specific microorganisms.¹⁷

Much research has been undertaken under in vitro and in vivo conditions to evaluate and compare the antimicrobial activity of endodontic irrigants at different concentrations. These studies most often used agar diffusion in addition to the direct contact method.¹⁸ Owing to differences in the experimental methodology and in the concentration of the test materials, conflicting results have been reported in the selection of ideal endodontic irrigates.¹⁹

Because some endodontic medicaments have undesirable side effects, the use of herbal compounds has become more common due to their anti-inflammatory, anti-oxidative, antibacterial, and antiviral effects. They are considered to be healthier and more environmentally acceptable.²⁰ They are easy to obtain and are well accepted by the host with many useful pharmacological actions comparable with synthetic drugs.

M. chamomilla, one of the most popular herbal extracts, is an annual plant from the Asteracea family.²¹

Goes et al²² studied the antimicrobial and anti-inflammatory properties of M. chamomilla, and found that it reduced biofilm accumulation in patients with gingival inflammation.

Venkataram et al²³ reported that application of a hydroalcoholic extract of chamomile resulted in effective and significant removal of the smear layer when compared with 2.5% sodium hypochlorite and effectively cleaned the coronal and middle thirds of the root canal.

Several studies have confirmed the antibacterial activity of M. chamomilla ethanolic extracts,^24–26 with the results varying depending on the tissue involved.^27–29 Shakya et al³⁰ found that M. chamomilla flowers significantly reduced E. faecalis growth. Plant extracts are well known as intracanal medicaments and irrigants for disinfection, and as agents for removal of the smear layer.³¹

Based on these previous investigations of various plant extracts, we selected a plant growing in our fields for antimicrobial evaluation.

In our study, the ethyl acetate extract of M. chamomilla applied alone was found to exert an inhibitory effect on E. faecalis, producing an average inhibitory zone of 9 mm. After combination with endodontic preparations, M. chamomilla was shown to be effective only after mixing with CHX, and the zone of inhibition was greater than that observed when CHX was used alone. After combination with sodium hypochlorite, M. chamomilla exerted no antibacterial effect against E. faecalis; no inhibition zone was observed.

Similar to our results, Shah et al³² showed that although Chamomile oil had more antimicrobial effect on Enterococcus faecalis than 2% CHX, both irrigators still had an observable effectiveness against E. faecalis.

According to the study of Jafari et al,³³ antibacterial effect of CHX and chamomile rapidly increased, thus indicating materials’ abilities to be adsorbed to dentine hydroxyapatite with prolonged gradual releases at therapeutic levels.

Previous research has proven that dentin powder has significantly lower inhibitory activity on medications, than human serum albumin,³⁴ even at high concentration.³⁵

The intensity of this inhibition is based on the drug concentration, the time of contact and the time of exposure of the bacteria to the mixture.³⁶

The Haapasalo model³⁷ is used to efficiently evaluate the interactions between root canal drugs, dentin, and microorganisms. Consistent with the results of Haapasalo et al, our study demonstrated that when endodontic solutions were mixed with dentin powder, the inhibitory areas decreased after contact with E. faecalis in comparison with samples without dentin powder at three time intervals. This can be explained by the fact that the components of dentin are responsible for the inhibitory effect in medications caused by moderation of the pH increase by the buffering effect of dentin.

Micro-organisms can use dentin as a source of nutrition using its collagen and calcium, and both of them may have a role in adherence capacity of micro-organisms to extracellular matrix proteins.³⁸

In our study, the results were recorded after 5 min, 60 min, and 24 h in order to evaluate inhibition over time. This change was more evident after the 24-h incubation period in samples treated with M. chamomilla after combination with chlorhexidine. Herbal extracts represent alternative endodontic therapy, so the results of this study may be considered as novelty.

Since, they are the few studies related to antimicrobial effect of M. chamomilla hydroalcoholic extracts in endodontic therapy, further studies should be conducted to evaluate the performance and antimicrobial activity of the above extract for clinical use as an intracanal medicament alone and in combination with other medicaments.

Conclusion

On the basis of the results of this study, we conclude that ethyl acetate extracts of M. chamomilla plant can be used as an alternative disinfectant for root canal disinfection alone, or in combination with CHX. M. chamomilla in combination with sodium hypochlorite showed no antibacterial effect.