In Vitro Antibacterial Effect of Pimpinella Anisum Essential Oil on Enterococcus Faecalis, Lactobacillus Casei, Actinomyces Naeslundii , and Aggregatibacter Actinomycetemcomitans

Introduction: Pimpinella anisum is a medicinal plant with antimicrobial, antifungal, and anti-oxidative properties. Limited studies have assessed the antibacterial properties of Pimpinella anisum on oral and dental pathogens. Aim: This in vitro study aimed to assess the antibacterial effect of Pimpinella anisum essential oil on Enterococcus faecalis , Lactobacillus casei , Actinomyces naeslundii , and Aggregatibacter actinomycetemcomitans . Materials and methods: After obtaining the essential oil of Pimpinella anisum , its antimicrobial activity was evaluated using the agar disc diffusion test. The minimum inhibitory concentration and minimum bactericidal concentration of the essential oil were also determined; 0.2% chlorhexidine was used as the positive control. Results: The mean diameter of growth inhibition zone was 39 mm for Enterococcus faecalis , 40 mm for Lactobacillus casei , 42 mm for Actinomyces naeslundii , and 18.5 mm for Aggregatibacter actinomycetemcomitans . The mean diameter of the growth inhibition zones for Enterococcus faecalis , Lactobacillus casei , and Actinomyces naeslundii was significantly greater than that of Aggregatibacter actinomy-cetemcomitans ( p =0.001). Also, the mean diameter of the growth inhibition zone of Actinomyces naeslundii was significantly larger than that of Enterococcus faecalis ( p =0.05) . The minimum inhibitory concentration and minimum bactericidal concentration of the essential oil for Enterococcus faecalis were 4.88% and 4.88%, respectively. These values were 9.76% and 9.76% for Lactobacillus casei , 9.76% and 4.88% for Actinomyces naeslundii , and 9.76% and 9.76% for Aggregatibacter actinomycetemcomitans , respectively. Conclusions: Pimpinella anisum essential oil was effective against all four microorganisms evaluated in this study. Since the lowest minimum inhibitory concentration and minimum bactericidal concentration were recorded for Enterococcus faecalis , this essential oil has maximum effects on Enterococcus faecalis . Future clinical studies are required to assess the antimicrobial efficacy of Pimpinella anisum essential oil in clinical samples.


INTRODUCTION
Oral and dental conditions are a public health dilemma. Many patients suffer from dental caries, periodontal disease and pulpal and periapical diseases caused by streptococci, actinomyces, and lactobacilli. [1,2] Streptococcus mutans plays an important role in initiation of caries. [3,4] Lactobacilli are responsible for caries progression especially in tooth crown. [5] Actinomyces are responsible for root caries, and are involved in periodontitis and root canal infection. [6,7] Enterococcus faecalis is responsible for root canal treatment failure, and development of treatment-resistant refractory infections. [8] Aggregatibacter actinomycetemcomitans is a major periodontal pathogen. [9,10] Since bacteria are the main causative agents of oral and dental conditions, elimination of pathogenic microorganisms is the best strategy for prevention of oral diseases. At present, use of medicinal plants as an alternative to synthetic medications has been gaining increasing popularity. [11] Use of mouthwashes, along with mechanical plaque removal methods, is suggested for elimination of oral pathogens. Scientists are in search of a medicinal plant to prevent dental plaque formation. An ideal medicinal plant for this purpose should not adversely affect the normal flora of the mouth, tooth colour, or sense of taste. Also, it should taste good and exert antibacterial effects comparable to the antibacterial effects of chlorhexidine (CHX). [12][13][14][15][16] Pimpinella anisum, also known as anise, is a one-year plant 1 m high, with narrow leaves, and good scent, which is extensively used in the Iranian traditional medicine. [17] Its essential oil contains anethole with antibacterial effects against Salmonella typhi, and Escherichia coli. It is used mainly for gastrointestinal diseases, but is also useful in the treatment of upper respiratory tract infections, fever, and oral and pharyngeal mucosal inflammation. [18] Its extract has antimicrobial properties against different bacteria and fungi. [19,20] Another study conducted in India showed that coconut oil can inhibit the proliferation of S. mutans similar to CHX. [1] In a study conducted in Turkey, the antioxidant and antimicrobial activities of aqueous and ethanolic extracts of P. anisum seed were proven. [5] Due to the increasing demand for use of herbal medicines as well as the optimal antimicrobial activity and low side effects of medicinal plants, researchers are attempting to synthesise herbal antimicrobial products against the pathogenic bacteria.

AIM
Considering the significance of this topic, this study aimed to assess the efficacy of the essential oil of P. anisum against E. faecalis, Lactobacillus casei, Actinomyces naeslundii, and A. actinomycetemcomitans in vitro.

Preparation of P. anisum essential oil
The essential oil of P. anisum was prepared by an expert person in the School of Pharmacy of Shahid Beheshti University of Medical Sciences. For preparation of the essential oil, 100 g of P. anisum was placed in an Erlenmeyer flask and 500 mL of distilled water was added to it. The essential oil was obtained using the Clevenger device for 4 hours. Averagely, 4 mL of essential oil was obtained from each 100 g of P. anisum. The primary concentration of the essential oil for antibacterial tests was considered to be 100%. The essential oil was then sterilized by using a 0.22µm microbiology filter.

Preparation of 0.5 McFarland standard concentrations
The bacteria were first cultured on brain heart infusion (BHI) agar and incubated at 37°C for 24 hours. Next, 0.5 McFarland [1.5×10 8 colony forming units (CFUs)/mL] bacterial suspensions were prepared for the agar disc diffusion test. For this purpose, 2 cc of sterile saline was added to each test tube and next, adequate amount of bacterial colonies from the 24-h culture was inoculated into the BHI agar. The contents of the tube were vortexed to obtain a homogenous suspension. The turbidity of the suspension was measured by a spectrophotometer at 600 nm wavelength; absorbance between 0.08-0.1 nm in this wavelength indicated 0.5 McFarland standard concentration.

Agar disc diffusion test
The agar disc diffusion test was used to assess the antimicrobial efficacy of the P. anisum essential oil. For this purpose, bacterial suspensions with 0.5 McFarland standard concentration were streak-cultured on BHI agar separately with sterile swabs. Each bacterial culture plate was divided into four regions. A blank disc dipped in 0.2% CHX was placed in one region as the positive control (0.2% CHX has proven antimicrobial effects and inhibits the bacterial growth; thus, a growth inhibition zone forms around the disc dipped in 0.2% CHX as the positive control). A blank disc was placed in another region as the negative control (the blank disc has no antimicrobial effect; thus, there would be no growth inhibition zone around it as the negative control), and discs containing P. anisum with primary concentration were placed on the remaining two regions.
Each test was repeated four times. Sterile gloves were worn during the entire experiment, and all procedures were performed next to the flame and under a hood. The plates containing A. actinomycetemcomitans and L. casei were placed in Gas-Pak anaerobic jars, respectively, for 48 hours. The plates containing E. faecalis and A. naeslundii were incubated in aerobic conditions at 37°C for 24 hours. After removing the plates from the incubator, a ruler was used to measure the diameter of the growth inhibition zone of the bacteria around the discs under a magnifier. The values were reported in millimetres.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
MIC is determined to assess the inhibitory effect of a material on proliferation of microorganisms. A 96-well enzyme-linked immunosorbent assay (ELISA) plate was used for the microdilution test.
Five rows (each row had 12 wells) of the plate were used in this test. For testing of the essential oil with three repetitions (the test instructions were the same for all bacteria), three rows were used. Also, one row was used as the positive and one row as the negative control. At first, 100 µL of the BHI broth was added to each well in all five rows. Next, for all three rows (row numbers 1, 2, and 3) that were related to testing of the essential oil with three repetitions, 100 µL of the primary concentration of the essential oil was added to number 1 to 12 wells of each of the three rows. Next, 100 µL of the contents of the first well was removed and added to the second well. This process was repeated until the 12 th well; 100 µL of the contents of the 12 th well was discarded. In this way, the essential oil was diluted by half each time it was transferred from one well to the other. In the next step, 10 µL of the bacterial suspension (0.5 McFarland standard concentration) was added to each well (#1 to 12). Also, one row (row C+) was considered as the positive control (100 µL of the BHI broth plus 10 µL of the bacterial suspension were added to wells 1 to 12) and another row (row C-) was considered as the negative control [only 100 µL of the BHI broth plus 100 µL of the primary concentration of the essential oil were added to these wells (#1 to 12), without any bacterial suspension].
Next, the ELISA plates of A. actinomycetemcomitans and L. casei were placed in Gas-Pak anaerobic jars for 48 hours. The plates containing E. faecalis and A. naeslundii were incubated in aerobic conditions at 37°C for 24 hours.
After incubation, the turbidity of the wells was measured by colorimetry. For this purpose, 0.01% resazurin was added to all wells and the plates were then placed in an incubator at 37°C. After 2 hours, the colour of the dye changed from blue to pink due to the activity of bacteria.
The first chromatic well was considered, and the previous wells with no colour change (lowest dilution in which no discolouration was observed) indicated the MIC of the essential oil for the respective microorganism as a fraction (percentage) of the primary concentration (100%).
To determine the MBC of the essential oil for the bacteria, samples were taken from one well before and 3 wells after the MIC well, as well as the MIC well itself. Then, the samples were passaged on the BHI agar.
Bacterial growth was evaluated after 24-48 hours of incubation as explained earlier. The MIC showing no growth on BHI agar indicated the MBC of the essential oil for the respective microorganism. The value was reported as a fraction (percentage) of the primary concentration (100%).

Statistical analysis
Data were analysed using SPSS software version 21 (SPSS Inc., IL, USA). The mean, standard deviation, minimum and maximum values were reported for the quantitative data. Independent t-test was used to compare the diameter of the growth inhibition zones of the studied bacteria around the essential oil and CHX discs, and two-way analysis of variance (ANOVA) was used to analyse the effect of the type of antimicrobial agent, type of microorganism, and the interaction effect of these two factors. P≤0.05 was considered statistically significant.

RESULTS
In the agar disc diffusion test, the diameter of the growth inhibition zone around the essential oil disc was maximum for A. naeslundii (mean value of 42 mm) followed by L. casei (40 mm), E. faecalis (39 mm), and A. actinomycetemcomitans (18.5 mm). The mean diameter of the growth inhibition zones for E. faecalis, L. casei, and A. naeslundii was significantly greater than that of A. actinomycetemcomitans (p=0.001). Also, the mean diameter of the growth inhibition zone of A. naeslundii was significantly larger than that of E. faecalis (p=0.05). Fig. 1 shows an agar disc diffusion test for E. faecalis. CHX disc was used as the positive control and a blank disc was used as the negative control. The difference in the diameter of the growth inhibition zone around the essential oil and CHX discs was significant for all bacteria (p<0.001), indicating that the essential oil was more potent than CHX against L. casei, A. naeslundii, and E. faecalis; while the antimicrobial efficacy of CHX against A. actinomycetemcomitans was higher than that of the essential oil (Table 1). No growth inhibition zone was noted around the blank disc.
In addition, the results of two-way ANOVA showed that the effect of the type of antimicrobial agent, type of microorganism, and the interaction effect of these two factors were significant on the diameter of the growth inhibition zone (p<0.001).
The MIC test showed that the essential oil was effective against all four microorganisms. The essential oil had maximum inhibitory effect on E. faecalis and A. naeslundii with MIC=4.88%. The MIC of the essential oil against A. actinomycetemcomitans and L. casei was 9.76%. Fig. 2 shows the results of MIC testing for A. actinomycetemcomitans after reaction with resazurin.
Regarding the MBC, the essential oil had maximum bactericidal effect on E. faecalis with MBC=4.88%. The MBC of the essential oil was 9.76% for L. casei, A. actinomycetemcomitans, and A. naeslundii .  MIC test results of A. actinomycetemcomitans after reaction with resazurin; rows 1, 2, and 3: for testing of the essential oil with three repetitions, BHI broth plus essential oil of P. anisum with bacterial suspension, negative control: BHI broth plus essential oil of P. anisum without any bacterial suspension, positive control: BHI broth plus bacterial suspension, blue colour: presence of bacteria without activity (the colour of resazurin is blue), red colour: presence of bacteria and their activity (the colour of resazurin changed from blue to red).

Fig. 3 shows the MBC test result for different concentrations of the essential oil against A. actinomycetemcomitans.
As shown, no colony grew in concentrations <1.1024.
According to both the MIC and MBC values, the essential oil had a maximum effect on E. faecalis. The MIC of the essential oil was 4.88% of the primary concentration (100%) for E. faecalis and A. naeslundii. The MIC of the essential oil for A. actinomycetemcomitans and L. casei was the same (9.76% of the primary concentration) (Fig. 4). Complete bacterial growth was noted in the positive control while no bacterial growth was noted in the negative control.

DISCUSSION
P. anisum has long been used in the Iranian traditional medicine as a disinfectant, antimicrobial, antifungal, antiviral, and antioxidant agent, muscle relaxant, and analgesic, anti-coagulative, and anti-inflammatory agent. [20] Considering the significance of treatment of oral infections and the side effects of chemical mouthwashes, this study aimed to assess the effect of P. anisum on oral pathogens. The results confirmed the inhibitory effect of P. anisum on four types of microorganisms. This effect was greater on E. faecalis, and A. naeslundii. According to the diameter of the growth inhibition zone, the essential oil of P. anisum had a smaller effect on A. actinomycetemcomitans compared with 0.2% CHX as the positive control. The reason for this finding may be the fact that A. actinomycetemcomitans is Gram-negative while the other tested bacteria are Gram-positive. Unlike Gram-positive bacteria, a lipopolysaccharide layer is present in the cell wall of Gram-negative bacteria. Thus, materials cannot easily pass through their cell wall, and only hydrophilic compounds with special structure can pass through it and affect the microorganism. Moreover, A. actinomycetemcomitans is a slow-growing microorganism and may show resistance to this essential oil, or might have protective mechanisms against it. On the other hand, L. casei was cultured in microaerophilic conditions and due to its slow growth, the essential oil had a smaller effect on it compared with E. faecalis and A. naeslundii. Evrendilek et al. evaluated the antimicrobial effects of the essential oils of 14 plant species including P. anisum on 10 microorganisms including four Gram-positive microorganisms such as Listeria innocua, coagulase negative staphylococcus, Bacillus subtilis, and S. aureus and six Gram-negative bacteria namely Proteus mirabilis, E. coli, Salmonella typhimurium, Salmonella enteritidis, Yersinia enterocolitica, and Klebsiella oxytoca by the agar disc diffusion method. They concluded that P. anisum had maximum antibacterial effects on Gram-positive bacteria such as S. aureus and Listeria. It had minimum antibacterial effects on Salmonella enteritidis and Proteus. Similarly to our study, they used the agar disc diffusion test, but did not assess the MIC and MBC. They evaluated a higher number of medicinal plants and also assessed the antimicrobial and anti-oxidative activity of the components by gas chromatography. They showed that P. anisum had significant antibacterial effects. [21] Kermanshah et al. in Iran evaluated the antibacterial effect of the hydroalcoholic extract of P. anisum, Satureja hortensis (savory) and Salvia officinalis (sage) on S. mutans, Lactobacillus rhamnosus, and Actinomyces viscosus using the agar disc diffusion test, and determined the MIC and MBC values as well. According to the obtained MIC value and the diameter of the growth inhibition zone, they concluded that all tested plant species had antibacterial activity of different levels. They also reported that the hydroalcoholic extract of anise had antibacterial effect on all three microorganisms. It had a maximum effect on S. mutans followed by L. rhamnosus. It had lower antibacterial activity against A. viscosus. In line with our findings, they confirmed the antibacterial effects of anise on S. mutans and L. rhamnosus. [19] Abu-Darvish et al. evaluated the antibacterial effects of the essential oil and hydroalcoholic extract of several medicinal plants including anise, thyme, and S. officinalis (sage) against S. aureus, Pseudomonas aeruginosa, and E. coli using the agar disc diffusion test. They reported that P. aeruginosa was resistant to the essential oil and hydroalcoholic extract of anise. However, the essential oil and hydroalcoholic extract of anise had mild antimicrobial effect on E. coli and moderate antimicrobial effect on S. aureus. Similarly to our study, they used the agar disc diffusion test; however, they used the essential oil and hydroalcoholic extract, and did not determine the MIC. Also, they used ciprofloxacin as the positive control. [22] Kermanshah et al. compared the antibacterial effect of the hydroalcoholic extract of S. officinalis (sage) and anise on S. mutans, L. rhamnosus, and A. viscosus in vitro by determining the MIC and MBC values. They reported that both herbs effectively inhibited the bacteria while S. officinalis (sage) had lower MIC and MBC compared with anise. They reported significant inhibitory effect of hydroalcoholic extract of anise on the bacteria. [18] Akhtar et al. in India evaluated the antimicrobial effects of the aqueous and alcoholic (acetone, methanolic, and ether) extracts of anise in vitro on four pathogens namely S. aureus, Streptococcus pyogenes, E. coli, and Klebsiella pneumonia. They concluded that the aqueous extract of anise was more effective than other extracts against the studied bacteria. The effect of methanolic extract of anise was lower than its aqueous extract. Acetone and ether extracts had no antibacterial effect. Ciprofloxacin served as the positive control and was more effective than all extracts. Their study had some differences with the present study. They used the aqueous form of the extract while we used its essential oil. They used ciprofloxacin as the positive control while we used CHX for this purpose. Moreover, they only performed the agar disc diffusion test while we performed MBC and MIC tests in addition to the agar disc diffusion test. They did not determine the MIC and MBC. They concluded that the aqueous extract of anise can serve as a cheap alternative to antibiotics. [23] Al-Bayati et al. in Iraq evaluated the antibacterial effect of alcoholic extract and essential oil of Thymus vulgaris and anise alone and in combination on E. coli, Proteus vulgaris, Proteus mirabilis, S. typhi, Klebsiella, P. aeruginosa, S. aureus, and Bacillus cereus by determining the MIC value, and concluded that the essential oil and alcoholic extract of these two plants had antibacterial effects against these pathogens. Thymus vulgaris was more effective than anise against all pathogens except for P. aeruginosa. However, the combination of both extracts was more effective than each of them alone against the pathogens particularly P. aeruginosa. Also, Gram-positive bacteria were more sensitive to the plant extracts compared with Gram-negative bacteria, except Proteus vulgaris. E. coli and Klebsiella pneumonia were resistant to P. anisum. Also it had minimum and maximum effects on S.
Folia Medica I 2022 I Vol. 64 I No. 5 typhi and S. aureus, respectively. They determined the MIC values similarly to our study but did not determine the MBC and did not use agar disc diffusion test. [24] Their results regarding optimal antibacterial efficacy of anise were in line with our findings. Tepe et al. in Turkey evaluated the anti-oxidative and antimicrobial activity of P. anisum and Pimpinella flabellifolia against 5 microorganisms namely Streptococcus pneumonia, B. cereus, Acinetobacter luufi, E. coli, and K. pneumonia and two fungi namely Candida albicans and Candida krusei using the agar disc diffusion test and MIC. P. anisum caused the maximum diameter of the growth inhibition zone in Acinetobacter luufi culture (18 mm) while Pimpinella flabellifolia caused the minimum diameter of the growth inhibition zone in E. coli culture (8 mm). The MIC of P. anisum and P. flabellifolia ranged from 9 to 72 mg/mL. The results showed that P. anisum and P. flabellifolia had moderate antibacterial effects on the tested microorganisms, and the antibacterial effect of P. anisum was stronger than that of P. flabellifolia. They assessed the antibacterial activity and anti-oxidative activity of two medicinal plants against several microorganisms. However, their results regarding the optimal antibacterial effect of P. anisum on the microorganisms were in agreement with our findings. [25] CONCLUSION Considering its optimal antibacterial activity, and fewer complications compared with chemical agents, it may be suitable for addition to toothpastes and root canal irrigating solutions. However, further studies on other properties, toxicity, and range of action of this essential oil against microorganisms are required prior to its use as a mouthwash.